scholarly journals Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

2021 ◽  
Vol 77 (10) ◽  
Author(s):  
Pieter De Bruyn ◽  
Maruša Prolič-Kalinšek ◽  
Alexandra Vandervelde ◽  
Milan Malfait ◽  
Yann G.-J. Sterckx ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Cell Parameter ◽  
Native Mass Spectrometry ◽  
Escherichia Coli O157 ◽  
Dna Binding Domain ◽  
Asymmetric Unit ◽  
Oligomeric Structure ◽  
Transcription Regulator ◽  
Acid Regulator

paaR2–paaA2–parE2 is a three-component toxin–antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix–turn–helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.

scholarly journals Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

2019 ◽  
Vol 476 (21) ◽  
pp. 3125-3139 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Jeannine Hess ◽  
Elen Shaw ◽  
Christina Spry ◽  
Robert Starley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Structural Biology ◽  
Ion Mobility ◽  
Biosynthetic Pathway ◽  
Screening Tool ◽  
Native Mass Spectrometry ◽  
Ion Mobility Mass Spectrometry ◽  
E Coli ◽  
Structural Insights

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

scholarly journals Thermodynamic Stability of the Transcription Regulator PaaR2 from Escherichia coli O157:H7

2019 ◽  
Vol 116 (8) ◽  
pp. 1420-1431 ◽  
Author(s):  
Pieter De Bruyn ◽  
San Hadži ◽  
Alexandra Vandervelde ◽  
Albert Konijnenberg ◽  
Maruša Prolič-Kalinšek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Thermodynamic Stability ◽  
Escherichia Coli O157 ◽  
Transcription Regulator

Bedeutung von Escherichia coli O157 beim Schlachtschaf in der Schweiz

2006 ◽  
Vol 148 (6) ◽  
pp. 289-295 ◽  
Author(s):  
C. Zweifel ◽  
M. Kaufmann ◽  
J. Blanco ◽  
R. Stephan
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157

Molecular profiling and antimicrobial susceptibility of Escherichia coli O157:H7 isolated in Bulgaria

2020 ◽  
Vol 23 (3) ◽  
pp. 310-318
Author(s):  
K. Koev ◽  
T. Stoyanchev ◽  
G. Zhelev ◽  
P. Marutsov ◽  
K. Gospodinova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Acid Sensitivity ◽  
Lower Extent ◽  
Phenotype Analysis ◽  
Anal Swab ◽  
Amoxicillin Clavulanic Acid ◽  
Primary Identification ◽  
Stx1 Gene ◽  
Cattle Farms

The purpose of this study was to detect the presence of shiga-toxin producing Escherichia coli (STEC) in faeces of healthy dairy cattle and to determine the sensitivity of isolates to several anti­microbial drugs. A total of 1,104 anal swab samples originating from 28 cattle farms were examined. After the primary identification, 30 strains were found to belong to serogroup О157. By means of conventional multiplex PCR, isolates were screened for presence of resistance genes stx1, stx2 and eaeА. Twenty-nine strains possesses amplicons with a size corresponding to genes stx2 and eaeA, one had amplicons also for the stx1 gene and one lacked amplicons of all three genes. Twenty-eight strains demonstrated amplicons equivalent to gene H7. The results from phenotype analysis of resistance showed preserved sensitivity to ceftriaxone, ceftazidime, cefotaxime, cephalothin, streptomycin, gentamicin, tetracycline, enrofloxacin and combinations sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid. Sensitivity to ampicillin was relatively preserved, although at a lower extent.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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