Journal: The Scientific World Journal

Article Title: High Level Aminoglycoside Resistance and Distribution of Aminoglycoside Resistant Genes among Clinical Isolates of Enterococcus Species in Chennai, India

doi: 10.1155/2014/329157

Figure Lengend Snippet: Results of high level aminoglycoside resistance and distribution of aminoglycoside modifying enzyme encoding genes among Enterococcus spp.

Article Snippet: This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

Techniques:

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: Poison-specific gyrase rescue mediated by QnrB1 protein. ( A ) Top: Domain architecture of E. coli DNA gyrase. Gyrase B is composed of ATPase, transducer (both purple) and TOPRIM (pink) domains. Indicated are molecular masses of different protein fragments (24, 43 and 47 kDa) produced and used in this work. Gyrase A is composed of winged-helix (WHD), tower, coiled-coil (all three blue) and β – pinwheel (cyan) domains. Below : A scheme of DNA gyrase mechanism. ( 1 ) A double-stranded DNA (G-segment (red)) is captured by the DNA gate, the ATPase gate is open to allow enter of the T-segment (yellow); ( 2 ) ATP binding leads to dimerization of the ATPase domains and trapping of the T-segment in the upper cavity; ( 3 ) this is followed by ATP hydrolysis, G-segment cleavage, DNA-gate opening and T-segment translocation. ( B ) Cartoon and molecular surface (transparent) representations of QnrB1 (PDB: 2XTW). Indicated are looped regions, previously found to be implicated in protective activity. ( C ). Plasmid supercoiling assay showing: lane 1: relaxed pBR322, lane 2: supercoiling by 1 U of gyrase, lane 3: lack of detectable nuclease activity in the purified QnrB1 (50 μM QnrB1) and lane 4: partial inhibition of gyrase by 50 μM QnrB1; lanes 6–13: gyrase inhibition by CFX (5 μM) and rescue by increasing concentrations of QnrB1 (0.04, 0.2, 1, 5, 10, 20, 25, 50 μM). Positions of negatively supercoiled and relaxed DNA are indicated by the graphics at the left (same notation used in other figures). ( D ). The gel shows gyrase supercoiling inhibition by increasing concentrations of albicidin in lanes 1–5 (0.016, 0.16, 1.6, 16, 160 μM). The effect of the addition of 5 μM QnrB1 to the same reactions (lanes 6–10).

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Produced, Binding Assay, Translocation Assay, Activity Assay, Plasmid Preparation, Purification, Inhibition

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: Measured MIC values. Measured MICs of albicidin (ALB), ciprofloxacin (CFX) and microcin B17 (MccB17) for E. coli BW25113 strain transformed with empty vector (pBAD) or plasmids expressing different PRPs

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Transformation Assay, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: QnrB1 decreases the amount of cleaved DNA. Gyrase cleavage assay shows that QnrB1 offers protection from cleavage induced by ciprofloxacin. ( A ) DNA gyrase (5 U) cleavage reactions with increasing amounts of ciprofloxacin run in the presence of ethidium bromide (EtBr). ( B ) DNA gyrase (5 U) cleavage reactions with increasing amounts of ciprofloxacin in presence of 5 μM QnrB1, run on an EtBr gel. Lane 1, relaxed pBR322. Lane 2, relaxed pBR322 with DNA gyrase. Lanes 3–11, relaxed pBR322 gyrase and increasing concentrations of ciprofloxacin (0.0016, 0.0008, 0.04, 0.2, 1, 5, 10, 15, 20 μM) ( C ) Cleaved DNA in the absence ( blue ) and presence ( red ) of QnrB1 quantified and plotted. Error bars represent standard deviation (SD) of 3 independent experiments.

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Cleavage Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: QnrB1 requires ATP hydrolysis to rescue DNA gyrase. ( A ) Time courses of DNA cleavage. The reactions contained 5 μM ciprofloxacin and 5 μM QnrB1 (as indicated) and were run without nucleotide, with ADPNP or ATP. After completion, the reactions were run on a gel without EtBr. ( B ) Same reactions run in the presence of EtBr. The amount of cleaved DNA is reduced when 5 μM QnrB1 is present and if ATP was added to the reaction. ( C ) Cleavage complex stability determined in a presence of 5 μM QnrB1. Initial DNA cleavage reactions were with 80 U of gyrase and 20 μM ciprofloxacin and were incubated for 10 min at 37°C to reach equilibrium and then diluted 20-fold with reaction buffer with or without 5 μM QnrB1. ( Top ) The samples were run on an EtBr gel. ( Bottom ) Linear DNA was quantified and plotted. Level of DNA cleavage at time 0 was set to 100%. Error bars represent the SD of at least three independent experiments.

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Incubation

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: QnrB1 stimulates gyrase ATPase activity. ( A ) ATPase rate data for 50 nM gyrase (A 2 B 2 ) complex mixed with: 10 nM DNA; 10 nM DNA and 5 μM QnrB1; 10 nM DNA, 5μM QnrB1 and 50 μM novobiocin; 5 μM QnrB1. ( B ) ATPase rate data for 4 μM GyrB43 mixed with QnrB1, drugs and DNA as indicated. DNA was used at 10 nM, novobiocin (Novo) at 50 μM, CFX at 5 μM and QnrB1 at 5 μM. ( C ) ATPase rate data and Michaelis-Menten fits for reactions conducted with different concentrations of ATP with ( blue ) or without ( red ) 5 μM QnrB1 for GyrB43. ( D ) ATPase assays data and Michaelis-Menten fit for reactions with 4 μM GyrB43 conducted with different concentrations of QnrB1 at constant concentration of ATP (1 mM). For all plots, error bars are expressed as the standard deviation of three independent experiments for the rate (mmol/min).

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Activity Assay, Concentration Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: Interactions between GyrB and QnrB1. ( A ) Fluorescence anisotropy assay showing binding of individual gyrase subunits and A 2 B 2 complex to QnrB1. Gray square – GyrA; red circle – GyrB; blue triangle – GyrB43; green triangle – gyrase A 2 B 2 complex. ΔmA indicates the change in anisotropy (in milliunits). Error bars represent the SD of three independent experiments. ( B ) In vitro pull-down assay with N-terminally FLAG-tagged QnrB1 and purified GyrA, GyrB and A 2 B 2 complex. Left – input, right – pull-down (eluates from M2 (α-FLAG) agarose). A Coomassie stained SDS-PAGE gel is shown. ( C ) A cartoon representation of QnrB1 monomer (grey, PDB: 2XTW) is shown with residues chosen for crosslinking experiments displayed as dark cyan cylinders. Residues that produce crosslinks when replaced with p Bpa are marked with arrows and displayed as magenta cylinders. ( D ) In vivo crosslinking anti-FLAG western blots of QnrB1 Q51, R77, Y123 and R167 p Bpa variants to chromosomally encoded GyrA-SPA and GyrB-SPA in E. coli . Visible band-shifts correspond to an increase in molecular weight of ∼25 kDa, roughly equivalent to QnrB1. ( E ) In vitro crosslinking anti-FLAG western blots of QnrB1 Q51, R77, Y123 and R167 p Bpa variants with different FLAG-tagged gyrase subunits and mixtures of both tagged and untagged subunits (GyrA/3xFLAG-GyrB or GyrA-FLAG/GyrB) (Af - C-terminally FLAG GyrA; Bf - N-terminally 3xFLAG GyrB). Crosslinks are indicated by an arrow.

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Fluorescence, Binding Assay, In Vitro, Pull Down Assay, Purification, Staining, SDS Page, In Vivo, Western Blot, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes

doi: 10.1093/nar/gkaa1266

Figure Lengend Snippet: Potential mechanism of action of QnrB1. Gyrase rescue from FQ by QnrB1: I . DNA gyrase cleavage complex formation. II . QnrB1 initial binding to the ATP-operated clamp (ATPase and transducer domains) in GyrB. III, IV . After ATP binding and hydrolysis, specific (loop-mediated) QnrB1 interaction results in fluoroquinolone removal and subsequent release of the PRP. This might be accompanied by the DNA release. QnrB1 gyrase inhibition : I . DNA gyrase with bound G segment. II . At high concentrations, QnrB1 competes with the T-segment and binds to the ATP-operated clamp, preventing T-segment binding. III, IV . After ATP hydrolysis, QnrB1 is released, which might be accompanied by the release of DNA.

Article Snippet: To measure the ability of DNA to compete with QnrB1, 50 nM Alexa-488-labeled QnrB1 was mixed with 1 μM gyrase and an increasing concentration of label free linearized pBR322 (Inspiralis).

Techniques: Binding Assay, Inhibition

Journal: PLoS ONE

Article Title: High-risk clones of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011–2012)

doi: 10.1371/journal.pone.0254805

Figure Lengend Snippet: Summary of the main findings.

Article Snippet: The characteristic combination bla CTX-M-15 , bla TEM-1B , bla OXA-1 , qnrB , and aac(6′)-Ib-cr was found in 8/19 isolates and 7/8 were also associated to IncF replicon group which was previously partially described in Morocco; bla CTX-M-15 , bla TEM-1b , bla OXA-1 , aac(6′)-Ib-cr , and qnrB1 genes were co-transferred and carried by a conjugative plasmid of high molecular weight [ ].

Techniques: Sampling, Clone Assay, CRISPR