A novel approach to study of the structural basis of enzyme polymorphism. Analysis of carboxylesterase B of Escherichia coli as model

In order to understand the structural basis of charge differences among enzyme variants without undertaking purification and sequencing of the protein, an original approach was developed. The approach is applicable to any enzyme or protein provided that there is a specific staining procedure. This consists, as a first step, in the projection of electrophoretically obtained mobility values versus pI of all variants into a two-dimensional profile. In a second step, starting from the most common variant, various theoretical possibilities of substitutions are envisaged, taking into consideration the pH of the electrophoretic conditions, pI of the variants and range of variations of the pK values of several amino acid side chains. In a third step, verification of the theoretical data is obtained through comparative protein titration curves by combined isoelectrofocusing-electrophoresis of several pairs of relevant variants. The validity of this approach is tested on the highly polymorphic carboxylesterase B enzyme of Escherichia coli and is found to provide valuable information.

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Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Scientific Reports ◽

10.1038/s41598-020-78640-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Robert J. Francis ◽

Gillian Robb ◽

Lee McCann ◽

Bhagwati Khatri ◽

James Keeble ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Three Dimensional ◽

Large Field ◽

Staining Procedure ◽

3D Analysis ◽

Mycobacterium Tuberculosis Infection ◽

In Vivo Models ◽

Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

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Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.10.2380 ◽

1996 ◽

Vol 40 (10) ◽

pp. 2380-2386 ◽

Cited By ~ 217

Author(s):

M J Everett ◽

Y F Jin ◽

V Ricci ◽

L J Piddock

Keyword(s):

Escherichia Coli ◽

Dna Sequences ◽

Quinolone Resistance ◽

Fluoroquinolone Resistance ◽

Polymorphism Analysis ◽

Single Mutation ◽

Wild Type ◽

Single Strand Conformational Polymorphism ◽

E Coli ◽

High Level

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

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A Novel Approach for Mutational Analysis of the 16s rRNA Molecule Using the Specialized Ribosome System in Escherichia Coli

Genetics of Translation ◽

10.1007/978-3-642-73139-6_27 ◽

1988 ◽

pp. 343-351

Author(s):

Herman A. de Boer ◽

Dennis H. Eaton ◽

Anna S. Hui

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Mutational Analysis ◽

Novel Approach ◽

Rrna Molecule

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Predictive Fluorescent Amplified-Fragment Length Polymorphism Analysis of Escherichia coli: High-Resolution Typing Method with Phylogenetic Significance

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1274-1279.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1274-1279 ◽

Cited By ~ 42

Author(s):

Catherine Arnold ◽

Lou Metherell ◽

Geraldine Willshaw ◽

Anthony Maggs ◽

John Stanley

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Amplified Fragment Length Polymorphism ◽

Polymorphism Analysis ◽

Enzyme Electrophoresis ◽

Typing Method ◽

E Coli

The fluorescent amplified-fragment length polymorphism (FAFLP) assay potentially amplifies a unique set of genome fragments from each bacterial clone. It uses stringently hybridizing primers which carry a fluorescent label. Precise fragment sizing is achieved by the inclusion of an internal size standard in every lane. Therefore, a unique genotype identifier(s) can be found in the form of fragments of precise size or sizes, and these can be generated reproducibly. In order to evaluate the potential of FAFLP as an epidemiological typing method with a valid phylogenetic basis, we applied it to 87 strains ofEscherichia coli. These comprised the EcoR collection, which has previously been classified by multilocus enzyme electrophoresis (MLEE) and which represents the genetic diversity of the species E. coli, plus 15 strains of the clinically important serogroup O157. FAFLP with an unlabelled nonselectiveEcoRI primer (Eco+0) and a labelled selectiveMseI primer (Mse+TA) gave strain-specific profiles. Fragments of identical sizes (in base pairs) were assumed to be identical, and the genetic distances between the strains were calculated. A phylogenetic tree derived from measure of distance correlated closely with the MLEE groupings of the EcoR collection and placed the verocytotoxin-producing O157 strains on an outlier branch. Our data indicate that FAFLP is suitable for epidemiological investigation of E. coli infection, providing well-defined and reproducible identifiers of genotype for each strain. Since FAFLP objectively samples the whole genome, each strain or isolate can be assigned a place within the broad context of the whole species and can also be subjected to a high-resolution comparison with closely related strains to investigate epidemiological clonality.

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The Integrity of α-β-α Sandwich Conformation Is Essential for a Novel Adjuvant TFPR1 to Maintain Its Adjuvanticity

Biomolecules ◽

10.3390/biom9120869 ◽

2019 ◽

Vol 9 (12) ◽

pp. 869 ◽

Cited By ~ 1

Author(s):

Qiao Li ◽

Xiuzhe Ning ◽

Yuepeng Wang ◽

Qing Zhu ◽

Yan Guo ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Peptide Vaccine ◽

Structural Basis ◽

Vaccine Adjuvant ◽

Novel Approach ◽

Immunologic Activity

TFPR1 is a novel peptide vaccine adjuvant we recently discovered. To define the structural basis and optimize its application as an adjuvant, we designed three different truncated fragments that have removed dominant B epitopes on TFPR1, and evaluated their capacity to activate bone marrow-derived dendritic cells and their adjuvanticity. Results demonstrated that the integrity of an α-β-α sandwich conformation is essential for TFPR1 to maintain its immunologic activity and adjuvanticity. We obtained a functional truncated fragment TFPR-ta ranging from 40–168 aa of triflin that has similar adjuvanticity as TFPR1 but with 2-log fold lower immunogenicity. These results demonstrated a novel approach to evaluate and improve the activity of protein-based vaccine adjuvant.

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Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

The Journal of Biochemistry ◽

10.1093/jb/mvz087 ◽

2019 ◽

Vol 167 (1) ◽

pp. 1-14

Author(s):

Koji Nagata ◽

Akitoshi Okada ◽

Jun Ohtsuka ◽

Takatoshi Ohkuri ◽

Yusuke Akama ◽

...

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Molecular Model ◽

Structural Basis ◽

Terminal Domain ◽

Helicase Dnab ◽

Accumulation Mechanism ◽

Interaction Domains ◽

Α Helix ◽

Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

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A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

Journal of Food Protection ◽

10.4315/0362-028x-72.7.1513 ◽

2009 ◽

Vol 72 (7) ◽

pp. 1513-1520 ◽

Cited By ~ 63

Author(s):

MANAN SHARMA ◽

DAVID T. INGRAM ◽

JITENDRA R. PATEL ◽

PATRICIA D. MILLNER ◽

XIAOLIN WANG ◽

...

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Plasmid Vector ◽

Green Fluorescent Protein Gene ◽

Escherichia Coli O157 ◽

E Coli ◽

Novel Approach ◽

Efficient Uptake ◽

Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

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Novel application of the matched case–control design to compare food supply chains during an Escherichia coli O157 outbreak, United Kingdom, 2016

Eurosurveillance ◽

10.2807/1560-7917.es.2018.23.18.17-00195 ◽

2018 ◽

Vol 23 (18) ◽

Cited By ~ 3

Author(s):

Thomas Inns ◽

Paul Cleary ◽

Nick Bundle ◽

Sarah Foulkes ◽

Ashley Sharp ◽

...

Keyword(s):

Escherichia Coli ◽

United Kingdom ◽

Distribution Network ◽

Case Control ◽

Food Supply Chains ◽

Novel Approach ◽

Food Borne ◽

Large Outbreak ◽

Matched Case ◽

Complex Distribution

There is a need for innovative methods to investigate outbreaks of food-borne infection linked to produce with a complex distribution network. The investigation of a large outbreak of Escherichia coli O157 PT34 infection in the United Kingdom in 2016 indicated that catering venues associated with multiple cases had used salad leaves sourced from one supplier. Our aim was to investigate whether catering venues linked to cases were more likely to have used salad leaves from this supplier. We conducted a matched case–control study, with catering venues as the units of analysis. We compared venues linked to cases to those without known linked cases. We included 43 study pairs and obtained information on salad leaf products received by each venue. The odds of a case venue being supplied with salad leaves by Supplier A were 7.67 times (95% confidence interval: 2.30–25.53) those of control venues. This association provided statistical evidence to support the findings of the other epidemiological investigations undertaken for this outbreak. This is a novel approach which is labour-intensive but which addresses the challenge of investigating exposures to food across a complex distribution network.

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