β lactamase activity  (Millipore)


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    Name:
    beta Lactamase
    Description:
    β Lactamase produced by bacteria is closely related to the penicillin binding proteins β Lactamase hydrolyzes β lactum antibiotics and is the prime cause of resistance development by bacteria There are four subclasses of β Lactamases Classes A C and D form an acyl enzyme via active site serine residue Class B β lactamases are metalloenzymes with zinc ion at their active site for β lactam hydrolysis Mutations in the β lactamases has resulted in the generation of extended spectrum β lactamases ESBLs As close to 900 types of β Lactamases are produced by microbes
    Catalog Number:
    l6170
    Price:
    None
    Applications:
    β--lactamase is used to inactivate β-lactam antibiotics by breaking open the β-lactam ring. β--lactamase is used to study antibiotic resistance and resistance suppression. Product L6170 is recombinantly produced based on the sequence of the enzyme from Pseudomonas aeruginosa, and is expressed in E. coli.
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    Structured Review

    Millipore β lactamase activity
    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and <t>β-lactamase</t>
    β Lactamase produced by bacteria is closely related to the penicillin binding proteins β Lactamase hydrolyzes β lactum antibiotics and is the prime cause of resistance development by bacteria There are four subclasses of β Lactamases Classes A C and D form an acyl enzyme via active site serine residue Class B β lactamases are metalloenzymes with zinc ion at their active site for β lactam hydrolysis Mutations in the β lactamases has resulted in the generation of extended spectrum β lactamases ESBLs As close to 900 types of β Lactamases are produced by microbes
    https://www.bioz.com/result/β lactamase activity/product/Millipore
    Average 95 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    β lactamase activity - by Bioz Stars, 2020-08
    95/100 stars

    Images

    1) Product Images from "Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria"

    Article Title: Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.72.1.880-889.2006

    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase
    Figure Legend Snippet: Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase

    Techniques Used: Incubation

    2) Product Images from "Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients"

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2007.05678.x

    Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.
    Figure Legend Snippet: Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Techniques Used: Activity Assay, Mutagenesis, Incubation

    3) Product Images from "Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis"

    Article Title: Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis

    Journal: mSystems

    doi: 10.1128/mSystems.00154-18

    β-Lactamase production and semiquantitative RT-PCR analysis of bla1 , bla2 , and 16S transcripts in B. anthracis strains. (A) β-Lactamase activity of culture supernatants from strains SK57 (PEN-R, rsiP 10 mutation), UT308 (PEN-R, sigP 183 mutation, rsiP 10 mutation), Ba3027 (PEN-S, rsiP 10 mutation), Sterne (PEN-S), and Ba0878 (PEN-S, wild-type strain) was measured using nitrocefin. Error bars represent averages ± standard deviations. **, P = 0.001 to 0.01 (statistical significance compared to β-lactamase-producing strain UT308); ***, P
    Figure Legend Snippet: β-Lactamase production and semiquantitative RT-PCR analysis of bla1 , bla2 , and 16S transcripts in B. anthracis strains. (A) β-Lactamase activity of culture supernatants from strains SK57 (PEN-R, rsiP 10 mutation), UT308 (PEN-R, sigP 183 mutation, rsiP 10 mutation), Ba3027 (PEN-S, rsiP 10 mutation), Sterne (PEN-S), and Ba0878 (PEN-S, wild-type strain) was measured using nitrocefin. Error bars represent averages ± standard deviations. **, P = 0.001 to 0.01 (statistical significance compared to β-lactamase-producing strain UT308); ***, P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activity Assay, Mutagenesis

    4) Product Images from "The Membrane-Proximal Tyrosine-Based Sorting Signal of Human Immunodeficiency Virus Type 1 gp41 Is Required for Optimal Viral Infectivity"

    Article Title: The Membrane-Proximal Tyrosine-Based Sorting Signal of Human Immunodeficiency Virus Type 1 gp41 Is Required for Optimal Viral Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.3.1069-1079.2004

    β-Lactamase-Vpr entry assay. Viruses were produced by cotransfection of the indicated provirus and pMM310, a β-lactamase-Vpr (BlaM-Vpr) fusion protein expression vector. Viruses were pelleted, resuspended in 1 ml of medium, normalized by p24 capsid concentration, and then used to infect P4.R5 cells for 5 h. After washing, cells were incubated with the β-lactamase-sensitive fluorochrome CCF2/AM for 16 to 18 h at 25°C. Analysis was performed by flow cytometry. Uncleaved substrate was detected as green fluorescence, and cleaved substrate was detected as blue fluorescence. (A) Plot of green fluorescence versus blue fluorescence. Loaded cell control refers to uninfected cells loaded with CCF2/AM. Box R3 encloses cells used for the ratio analysis in panel B, cells positive for CCF2/AM loading. (B) Data are expressed as a ratio of blue to green fluorescence versus side scatter and are gated using uninfected cells (loaded cell control). AMD3100 (100 nm) is a CXCR4 antagonist used to block entry into cells.
    Figure Legend Snippet: β-Lactamase-Vpr entry assay. Viruses were produced by cotransfection of the indicated provirus and pMM310, a β-lactamase-Vpr (BlaM-Vpr) fusion protein expression vector. Viruses were pelleted, resuspended in 1 ml of medium, normalized by p24 capsid concentration, and then used to infect P4.R5 cells for 5 h. After washing, cells were incubated with the β-lactamase-sensitive fluorochrome CCF2/AM for 16 to 18 h at 25°C. Analysis was performed by flow cytometry. Uncleaved substrate was detected as green fluorescence, and cleaved substrate was detected as blue fluorescence. (A) Plot of green fluorescence versus blue fluorescence. Loaded cell control refers to uninfected cells loaded with CCF2/AM. Box R3 encloses cells used for the ratio analysis in panel B, cells positive for CCF2/AM loading. (B) Data are expressed as a ratio of blue to green fluorescence versus side scatter and are gated using uninfected cells (loaded cell control). AMD3100 (100 nm) is a CXCR4 antagonist used to block entry into cells.

    Techniques Used: Produced, Cotransfection, Expressing, Plasmid Preparation, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Fluorescence, Blocking Assay

    5) Product Images from "Effect of β-Lactamase inhibitors on in vitro activity of β-Lactam antibiotics against Burkholderia cepacia complex species"

    Article Title: Effect of β-Lactamase inhibitors on in vitro activity of β-Lactam antibiotics against Burkholderia cepacia complex species

    Journal: Antimicrobial Resistance and Infection Control

    doi: 10.1186/s13756-016-0142-3

    β-lactamase activity and corresponding MIC values for a selection of Bcc strains tested. Dark grey bars: β-lactamase activity relative to untreated controls, light grey bars: MIC values (mg/L) for a ) B. multivorans LMG 18825, b ) B. arboris R-132, c ) B. cenocepacia LMG 16656 and d ) B. vietnamiensis LMG 18835. * represents statistically significant differences compared to treatment with the β-lactam antibiotic alone ( p
    Figure Legend Snippet: β-lactamase activity and corresponding MIC values for a selection of Bcc strains tested. Dark grey bars: β-lactamase activity relative to untreated controls, light grey bars: MIC values (mg/L) for a ) B. multivorans LMG 18825, b ) B. arboris R-132, c ) B. cenocepacia LMG 16656 and d ) B. vietnamiensis LMG 18835. * represents statistically significant differences compared to treatment with the β-lactam antibiotic alone ( p

    Techniques Used: Activity Assay, Selection

    6) Product Images from "Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome"

    Article Title: Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0186920

    Investigation of secretion and translocation of antitoxins and chaperones. ( A ) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture OD 600 of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kDa): Orf182-bla (140), Orf48-bla (41), CcdA-bla (38), MvpA-bla (38), Spa15-bla (44), IpgA-bla (44). ( B ) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of A 486nm . e.M.u.: equivalent Miller unit. Data are from 4 independent experiments. ( C ) CCF2-AM-loaded Jurkat T cells were infected with WT strains for 1 hour. Translocated cells were detected by flow cytometry. Data are from 3 independent experiments. ( B - C ) **p
    Figure Legend Snippet: Investigation of secretion and translocation of antitoxins and chaperones. ( A ) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture OD 600 of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kDa): Orf182-bla (140), Orf48-bla (41), CcdA-bla (38), MvpA-bla (38), Spa15-bla (44), IpgA-bla (44). ( B ) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of A 486nm . e.M.u.: equivalent Miller unit. Data are from 4 independent experiments. ( C ) CCF2-AM-loaded Jurkat T cells were infected with WT strains for 1 hour. Translocated cells were detected by flow cytometry. Data are from 3 independent experiments. ( B - C ) **p

    Techniques Used: Translocation Assay, Molecular Weight, Expressing, Incubation, Activity Assay, Infection, Flow Cytometry, Cytometry

    Experimental confirmation of secretion and translocation of MS hits. ( A ) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture optical density at 600 nm (OD 600 ) of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kiloDaltons, kDa): Orf182-bla (140), OspI-bla (53), Orf86-bla (40), Orf48-bla (41), Orf176-bla (40), Orf13-bla (51), Orf131a-bla (38). ( B ) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of absorbance at 486 nm (A 486 ). e.M.u.: equivalent Miller unit. Data are from 3 independent experiments. *p
    Figure Legend Snippet: Experimental confirmation of secretion and translocation of MS hits. ( A ) ipaD Shigella lysates were analysed by immunoblotting with anti-β-lactamase antibody. Load equivalent to a bacterial culture optical density at 600 nm (OD 600 ) of 0.2 for each lane. Chimeric proteins tested and their expected molecular weight (kiloDaltons, kDa): Orf182-bla (140), OspI-bla (53), Orf86-bla (40), Orf48-bla (41), Orf176-bla (40), Orf13-bla (51), Orf131a-bla (38). ( B ) Supernatants of ipaD strains expressing the Orf-bla chimeric proteins were incubated with nitrocefin. Enzymatic activity was calculated based on measurement of absorbance at 486 nm (A 486 ). e.M.u.: equivalent Miller unit. Data are from 3 independent experiments. *p

    Techniques Used: Translocation Assay, Mass Spectrometry, Molecular Weight, Expressing, Incubation, Activity Assay

    7) Product Images from "Staphylococcus aureus Extracellular Vesicles Carry Biologically Active ?-Lactamase"

    Article Title: Staphylococcus aureus Extracellular Vesicles Carry Biologically Active ?-Lactamase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00522-12

    Enzymatic activities of S. aureus soluble and EV-associated BlaZ were similar. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ (0.002 to 0.2 μg) and EVs (1.0 μg) detected with lab-made polyclonal anti-BlaZ antibodies. The molecular mass standard is indicated on the left (in kDa). Note that 1.0 μg of S. aureus EVs contained 0.02 ± 0.001 μg of BlaZ. (B) β-Lactamase activities of the purified S. aureus ATCC 14458 soluble BlaZ (0.02 μg/ml) and EVs (1.0 μg/ml). The activities were the initial rates of hydrolysis of penicillin G (1 μM) for 15 min and expressed in change of absorbance units/min (Δ A 240 /min). ***, P
    Figure Legend Snippet: Enzymatic activities of S. aureus soluble and EV-associated BlaZ were similar. (A) Western blotting of the purified S. aureus ATCC 14458 soluble BlaZ (0.002 to 0.2 μg) and EVs (1.0 μg) detected with lab-made polyclonal anti-BlaZ antibodies. The molecular mass standard is indicated on the left (in kDa). Note that 1.0 μg of S. aureus EVs contained 0.02 ± 0.001 μg of BlaZ. (B) β-Lactamase activities of the purified S. aureus ATCC 14458 soluble BlaZ (0.02 μg/ml) and EVs (1.0 μg/ml). The activities were the initial rates of hydrolysis of penicillin G (1 μM) for 15 min and expressed in change of absorbance units/min (Δ A 240 /min). ***, P

    Techniques Used: Western Blot, Purification

    8) Product Images from "Antibiotic Trapping by Plasmid-Encoded CMY-2 ?-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli"

    Article Title: Antibiotic Trapping by Plasmid-Encoded CMY-2 ?-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02459-12

    Zymogram and SDS-PAGE gel revealing expression of β-lactamase in the carbapenem-resistant E. coli isolates. (A) The zymogram was obtained by incubating the gel, on which periplasmic proteins from isolates EC-8, EC-9, EC-2, and EC-3 were separated,
    Figure Legend Snippet: Zymogram and SDS-PAGE gel revealing expression of β-lactamase in the carbapenem-resistant E. coli isolates. (A) The zymogram was obtained by incubating the gel, on which periplasmic proteins from isolates EC-8, EC-9, EC-2, and EC-3 were separated,

    Techniques Used: SDS Page, Expressing

    Covalent modification of CMY-2 β-lactamase in vivo and stability of the complex. (A) Cells of strain BL21(DE3) and its porin-deficient mutant, CE1536, both carrying the CMY-2-encoding plasmid, were incubated for 20 min with meropenem at the concentrations
    Figure Legend Snippet: Covalent modification of CMY-2 β-lactamase in vivo and stability of the complex. (A) Cells of strain BL21(DE3) and its porin-deficient mutant, CE1536, both carrying the CMY-2-encoding plasmid, were incubated for 20 min with meropenem at the concentrations

    Techniques Used: Modification, In Vivo, Mutagenesis, Plasmid Preparation, Incubation

    Inhibition of β-lactamase activity by meropenem. Periplasmic extracts of isolate EC-8 were incubated for 1 min with various concentrations of meropenem as indicated. Subsequently, the remaining β-lactamase activity was determined using
    Figure Legend Snippet: Inhibition of β-lactamase activity by meropenem. Periplasmic extracts of isolate EC-8 were incubated for 1 min with various concentrations of meropenem as indicated. Subsequently, the remaining β-lactamase activity was determined using

    Techniques Used: Inhibition, Activity Assay, Incubation

    9) Product Images from "Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase"

    Article Title: Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142870

    Membrane topology of the E . coli BacA protein. This topological model was determined experimentally as described in Materials and Methods by using the β-lactamase as a reporter of the transmembrane topology of BacA. P1-P3: periplasmic domains; C1-C3: cytoplasmic domains. Transmembrane segments were predicted by the TMAP program [ 25 , 26 ]. BacA residues colored in red and blue are invariant and highly-conserved residues, respectively. Stars indicate the residues that were used as junction points for the construction of the different BacA-BlaM hybrid proteins. Blue and green stars correspond to hybrid proteins that conferred and did not confer ampicillin resistance to cells when plated at a low cell density, respectively. The sequences of the two characteristic BacA motifs (BacA1 and BacA2) are shown in the two boxes (invariant residues are in capitals, h correspond to hydrophobic residues and x to any residue; numbers in superscript indicate the position of these residues in the E . coli BacA protein sequence).
    Figure Legend Snippet: Membrane topology of the E . coli BacA protein. This topological model was determined experimentally as described in Materials and Methods by using the β-lactamase as a reporter of the transmembrane topology of BacA. P1-P3: periplasmic domains; C1-C3: cytoplasmic domains. Transmembrane segments were predicted by the TMAP program [ 25 , 26 ]. BacA residues colored in red and blue are invariant and highly-conserved residues, respectively. Stars indicate the residues that were used as junction points for the construction of the different BacA-BlaM hybrid proteins. Blue and green stars correspond to hybrid proteins that conferred and did not confer ampicillin resistance to cells when plated at a low cell density, respectively. The sequences of the two characteristic BacA motifs (BacA1 and BacA2) are shown in the two boxes (invariant residues are in capitals, h correspond to hydrophobic residues and x to any residue; numbers in superscript indicate the position of these residues in the E . coli BacA protein sequence).

    Techniques Used: Sequencing

    10) Product Images from "Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients"

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2007.05678.x

    Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.
    Figure Legend Snippet: Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Techniques Used: Activity Assay, Mutagenesis, Incubation

    11) Product Images from "Horizontal Transfer of blaCMY-Bearing Plasmids among Clinical Escherichia coli and Klebsiella pneumoniae Isolates and Emergence of Cefepime-Hydrolyzing CMY-19"

    Article Title: Horizontal Transfer of blaCMY-Bearing Plasmids among Clinical Escherichia coli and Klebsiella pneumoniae Isolates and Emergence of Cefepime-Hydrolyzing CMY-19

    Journal:

    doi: 10.1128/AAC.50.2.534-541.2006

    Plasmid profiles and Southern hybridization. (A) Plasmid profiles of clinical isolates and their tranconjugants; (B) hybridization with the probe specific for the CMY-1- and MOX-1-type β-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1,
    Figure Legend Snippet: Plasmid profiles and Southern hybridization. (A) Plasmid profiles of clinical isolates and their tranconjugants; (B) hybridization with the probe specific for the CMY-1- and MOX-1-type β-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1,

    Techniques Used: Plasmid Preparation, Hybridization, Marker

    Plasmid patterns after restriction enzyme digestion and Southern hybridization. (A) SacI-digested plasmid DNAs prepared from the representative transconjugants; (B) hybridization patterns with the probe specific for CMY-1- and MOX-1-type β-lactamase
    Figure Legend Snippet: Plasmid patterns after restriction enzyme digestion and Southern hybridization. (A) SacI-digested plasmid DNAs prepared from the representative transconjugants; (B) hybridization patterns with the probe specific for CMY-1- and MOX-1-type β-lactamase

    Techniques Used: Plasmid Preparation, Hybridization

    12) Product Images from "Genetic and Biochemical Characterization of TRU-1, the Endogenous Class C ?-Lactamase from Aeromonas enteropelogenes ▿"

    Article Title: Genetic and Biochemical Characterization of TRU-1, the Endogenous Class C ?-Lactamase from Aeromonas enteropelogenes ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01252-09

    Unrooted tree showing the relationship between TRU-1 and representative chromosomal and plasmid-encoded AmpC class C β-lactamases. The Aeromonas class C β-lactamase (BL) cluster includes the chromosome-encoded enzymes of A. punctata P239
    Figure Legend Snippet: Unrooted tree showing the relationship between TRU-1 and representative chromosomal and plasmid-encoded AmpC class C β-lactamases. The Aeromonas class C β-lactamase (BL) cluster includes the chromosome-encoded enzymes of A. punctata P239

    Techniques Used: Plasmid Preparation

    Purification and biochemical characterization of the TRU-1 β-lactamase.
    Figure Legend Snippet: Purification and biochemical characterization of the TRU-1 β-lactamase.

    Techniques Used: Purification

    13) Product Images from "The importance of the trans-enamine intermediate as a ?-lactamase inhibition strategy probed in inhibitor-resistant SHV ?-lactamase variants"

    Article Title: The importance of the trans-enamine intermediate as a ?-lactamase inhibition strategy probed in inhibitor-resistant SHV ?-lactamase variants

    Journal: ChemMedChem

    doi: 10.1002/cmdc.201200006

    SA2-13 and its active site interactions when bound to SHV-1 S130G (A) Chemical structure of SA2-13. (B) Electron density of the SA2-13 compound in the active site of S130G SHV-1 β-lactamase. Unbiased omit F o- F c map is contoured at 2.5σ. SA2-13 carbon atoms are colored black and nitrogen, oxygen, and sulfur atoms in different shades of gray. (C) Stereo view of interactions of SA2-13 within S130G SHV-1 β-lactamase active site. Dashed black lines indicated hydrogen bonds. Water molecules are also depicted as spheres.
    Figure Legend Snippet: SA2-13 and its active site interactions when bound to SHV-1 S130G (A) Chemical structure of SA2-13. (B) Electron density of the SA2-13 compound in the active site of S130G SHV-1 β-lactamase. Unbiased omit F o- F c map is contoured at 2.5σ. SA2-13 carbon atoms are colored black and nitrogen, oxygen, and sulfur atoms in different shades of gray. (C) Stereo view of interactions of SA2-13 within S130G SHV-1 β-lactamase active site. Dashed black lines indicated hydrogen bonds. Water molecules are also depicted as spheres.

    Techniques Used:

    14) Product Images from "ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides"

    Article Title: ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.221960

    Key residues in the ClbP active site. A , residues of ClbP located in close vicinity of the binding site. Hydrogen bonds are indicated by pink dashed lines , and the water molecules are indicated by W . Carbon atoms are colored green , oxygen atoms are colored red, and nitrogen atoms are colored blue. B , overlay of penicillin-recognizing motifs of ClbP, Pab87 peptidase, and AmpC β-lactamase. Carbon atoms of ClbP, Pab87 peptidase, and AmpC β-lactamase are in green , yellow , and gray , respectively. The motifs S xx K, Y x (N/S/T), and (K/H)(S/T/G)G are numbered in red , blue , and green , respectively, and residue numbering is according to the ClbP sequence. C and D , phenotypic analysis of pks island activity in E. coli DH10B/pBAC pks deleted or not for the clbP gene and trans-complemented with ClbP WT protein or the S95A, K98T, or Y186G active site ClbP mutants. HeLa cells were infected 4 h with 100 E. coli per cell, and then the cells were washed and incubated with gentamicin for 4–72 h. C , anti-5His Western blot analysis of the expression of His-tagged WT and active site ClbP mutants in E. coli DH10B/pBAC pks Δ clbP after infection. D , host histone H2AX Ser-139 phosphorylation (γH2AX) indicative of DNA double-strand breaks was assayed by confocal immunofluorescence 4 h after infection. DNA and γH2AX are pseudocolored in blue and red , respectively ( bars , 20 μm). Cell swelling (megalocytosis) was observed following Giemsa staining 72 h after infection ( bars , 50 μm). G 2 cell cycle arrest following DNA damage was assessed by flow cytometric DNA content analysis, 48 h after infection.
    Figure Legend Snippet: Key residues in the ClbP active site. A , residues of ClbP located in close vicinity of the binding site. Hydrogen bonds are indicated by pink dashed lines , and the water molecules are indicated by W . Carbon atoms are colored green , oxygen atoms are colored red, and nitrogen atoms are colored blue. B , overlay of penicillin-recognizing motifs of ClbP, Pab87 peptidase, and AmpC β-lactamase. Carbon atoms of ClbP, Pab87 peptidase, and AmpC β-lactamase are in green , yellow , and gray , respectively. The motifs S xx K, Y x (N/S/T), and (K/H)(S/T/G)G are numbered in red , blue , and green , respectively, and residue numbering is according to the ClbP sequence. C and D , phenotypic analysis of pks island activity in E. coli DH10B/pBAC pks deleted or not for the clbP gene and trans-complemented with ClbP WT protein or the S95A, K98T, or Y186G active site ClbP mutants. HeLa cells were infected 4 h with 100 E. coli per cell, and then the cells were washed and incubated with gentamicin for 4–72 h. C , anti-5His Western blot analysis of the expression of His-tagged WT and active site ClbP mutants in E. coli DH10B/pBAC pks Δ clbP after infection. D , host histone H2AX Ser-139 phosphorylation (γH2AX) indicative of DNA double-strand breaks was assayed by confocal immunofluorescence 4 h after infection. DNA and γH2AX are pseudocolored in blue and red , respectively ( bars , 20 μm). Cell swelling (megalocytosis) was observed following Giemsa staining 72 h after infection ( bars , 50 μm). G 2 cell cycle arrest following DNA damage was assessed by flow cytometric DNA content analysis, 48 h after infection.

    Techniques Used: Binding Assay, Sequencing, Activity Assay, Infection, Incubation, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry

    Related Articles

    Clone Assay:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

    Flow Cytometry:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Amplification:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Fluorescence:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Beta Lactamase Activity Assay:

    Article Title: Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis
    Article Snippet: .. Broth cultures in the late-exponential-growth phase (105 CFU/ml) of B. anthracis strains SK57, Ba3027, Ba0878, Sterne (PEN-S, select agent-excluded strain), and UT308 (PEN-R, select agent-excluded derivative of strain 32) were tested for β-lactamase activity using a nitrocefin-based quantitative β-lactamase activity assay (β-lactamase activity assay kit, MAK221; Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. .. Supernatants from 500 µl of culture from each strain were collected by centrifugation at 8,000 × g for 2 min through a 0.1-μm-pore-size polyvinylidene difluoride (PVDF) Ultrafree-MC spin-filter column (Millipore, Billerica, MA, USA).

    Cytometry:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Purification:

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
    Article Snippet: .. The quality of the purified periplasmic fractions was tested by Western blot assay using anti-GroEL (kindly donated by Mario Cancino) and anti-β-lactamase (Chemicon, Temecula, CA) antibodies to detect GroEL (a cytoplasmic protein) and β-lactamase (a periplasmic protein), respectively. ..

    Produced:

    Article Title: Combinatorial microfluidic droplet engineering for biomimetic material synthesis
    Article Snippet: .. Soluble bacterial extracts, including a gene coding for β-lactamase, were used as the internal water phase ( , fig. S10, and movie S5). β-Lactamase was selected as an enzyme reporter rather than inherently fluorescent proteins to take advantage of the potential for fluorescence signal amplification: β-lactamase cleavage of a fluorogenic substrate (“CCF2,” Sigma; emission, 518 nm) generates a large excess of fluorescent small-molecule product (emission, 447 nm) relative to the number of β-lactamase proteins produced within the droplets, thus helping ensure that β-lactamase production within droplets can be measured using a commercial flow cytometer. ..

    Concentration Assay:

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein
    Article Snippet: .. The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.). ..

    Incubation:

    Article Title: Binding Properties of a Peptide Derived from ?-Lactamase Inhibitory Protein
    Article Snippet: .. The β-lactamase gene was induced by adding isopropyl-β- d -thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubation at 25°C for 4 h. Following induction the cells were pelleted and the supernatant containing the secreted, soluble β-lactamase was concentrated to 100 ml with an Amicon Centriprep-10 concentrator (Millipore Corp.). ..

    Activity Assay:

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients
    Article Snippet: .. To assay β-lactamase activity in culture supernatants, P. aeruginosa cultures grown overnight with LB broth were passed through a filter (0.22 µm pore size, Millipore), and the cell-free supernatants were assayed for β-lactamase activity 30 min after addition of nitrocefin (Calbiochem), as described by the manufacturer, except that A490 was measured, and corrected by subtracting the value measured for the supernatant without nitrocefin. .. To determine the proportion of β-lactamase activity that is cell-associated, P. aeruginosa cultures grown overnight with LB broth were diluted 1:100 in LB broth and grown for 12 h. These cultures were centrifuged and the supernatant saved.

    Article Title: Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis
    Article Snippet: .. Broth cultures in the late-exponential-growth phase (105 CFU/ml) of B. anthracis strains SK57, Ba3027, Ba0878, Sterne (PEN-S, select agent-excluded strain), and UT308 (PEN-R, select agent-excluded derivative of strain 32) were tested for β-lactamase activity using a nitrocefin-based quantitative β-lactamase activity assay (β-lactamase activity assay kit, MAK221; Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions. .. Supernatants from 500 µl of culture from each strain were collected by centrifugation at 8,000 × g for 2 min through a 0.1-μm-pore-size polyvinylidene difluoride (PVDF) Ultrafree-MC spin-filter column (Millipore, Billerica, MA, USA).

    Article Title: Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria
    Article Snippet: .. After 60 min, GEM particles and insoluble amylose azure were spun down, and the absorbance at 595 nm was determined. β-Lactamase activity was measured by adding 40 μl nitrocefin (CalBiochem) to GEM particles loaded with β-PA in 1 ml (final volume) of PBS. .. For immunofluorescence microscopy, suspensions containing 100 μl of GEM particles incubated with a PA fusion were washed twice with demineralized water and resuspended in an equal volume of PBS containing 1% BSA and mouse anti-c-myc antibody that was diluted 1:50.

    Expressing:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

    Western Blot:

    Article Title: VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli
    Article Snippet: .. The quality of the purified periplasmic fractions was tested by Western blot assay using anti-GroEL (kindly donated by Mario Cancino) and anti-β-lactamase (Chemicon, Temecula, CA) antibodies to detect GroEL (a cytoplasmic protein) and β-lactamase (a periplasmic protein), respectively. ..

    Plasmid Preparation:

    Article Title: Inhibition of the Class C ?-Lactamase from Acinetobacter spp: Insights into Effective Inhibitor Design †
    Article Snippet: .. For large-scale protein expression and β-lactamase characterization, the bla ADC gene (specifically, bla ADC-7 ) was cloned into pET24a (+) vector (kanamycin resistant, Novagen, Madison, WI) following a previously published method ( ). .. After sequencing verification, the correct construct was maintained in E. coli DH10B cells and transformed into E. coli BL21(DE3) cells for protein expression.

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    Millipore β lactamase activity
    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and <t>β-lactamase</t>
    β Lactamase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore lactamase activity
    Reporter assay for detection of AmpG permease activity. The <t>β-lactamase</t> assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.
    Lactamase Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase

    Journal: Applied and Environmental Microbiology

    Article Title: Novel Surface Display System for Proteins on Non-Genetically Modified Gram-Positive Bacteria

    doi: 10.1128/AEM.72.1.880-889.2006

    Figure Lengend Snippet: Immobilization of enzymes on the surface of lactococcal GEM particles. (a) Relative α-amylase activities on GEM particles incubated with culture medium containing soluble AmyL, PA (PA3), or α-PA. (b) Relative α-amylase and β-lactamase

    Article Snippet: After 60 min, GEM particles and insoluble amylose azure were spun down, and the absorbance at 595 nm was determined. β-Lactamase activity was measured by adding 40 μl nitrocefin (CalBiochem) to GEM particles loaded with β-PA in 1 ml (final volume) of PBS.

    Techniques: Incubation

    Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Journal: Molecular microbiology

    Article Title: Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients

    doi: 10.1111/j.1365-2958.2007.05678.x

    Figure Lengend Snippet: Inactivation of lasR confers increased β-lactamase activity and β-lactam tolerance. A. Ceftazidime-resistant colonies emerged in a lawn of CF416 or CF416L1 cells on LB agar with 20 µg ml −1 of ceftazidime (Petri dish upper and lower halves), but only the lasR mutant lawns yielded small partially resistant colonies whose growth is inhibited by the addition of 8 µg ml −1 of the β-lactamase inhibitor tazobactam (Petri dish lower halves). Photographs were taken after incubation overnight at 37°C followed by 3 days at 22°C. B. β-Lactamase activity in culture supernatants, measured as a change in optical density at 490 nm (ΔA 490 ) after addition of the chromogenic substrate nitrocefin. Cases with no change are marked with an asterisk. Values are the average of three technical replicates, and error bars show standard deviations. Equivalent results were obtained using a qualitative whole-culture assay. C. β-Lactamase activity in whole cultures of CF416 and CF416 lasR ::Gm. Values are the averages of three cultures, and error bars show standard error of the mean. One unit of β-lactamase is defined as 1 µmol of nitrocefin hydrolysed per min per mg of total protein. D. Reduced killing by ceftazidime of the lasR mutant CF416L1 relative to CF416 and CF416L1R. Values are the average cfu of two cultures, and error bars show standard deviations.

    Article Snippet: To assay β-lactamase activity in culture supernatants, P. aeruginosa cultures grown overnight with LB broth were passed through a filter (0.22 µm pore size, Millipore), and the cell-free supernatants were assayed for β-lactamase activity 30 min after addition of nitrocefin (Calbiochem), as described by the manufacturer, except that A490 was measured, and corrected by subtracting the value measured for the supernatant without nitrocefin.

    Techniques: Activity Assay, Mutagenesis, Incubation

    Schematic of the Tlpp function and its upregulation in MRSA after β-lactam treatment.

    Journal: bioRxiv

    Article Title: β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

    doi: 10.1101/353227

    Figure Lengend Snippet: Schematic of the Tlpp function and its upregulation in MRSA after β-lactam treatment.

    Article Snippet: Preparation of the total bacteria proteins and the culture supernatant proteinsOvernight MRSA culture was diluted 1:100 in BHI medium with or without the addition of β-lactam antibiotics, cultivated at 37 °C to an optical density (OD) at 600 nm of 2.0.

    Techniques:

    SarA bound to the tlpp cluster promoter region to control the β-lactam-stimulated Tlpp expression in MRSA. (A) β-galactosidase assay. The pOS1- tlpps P reporter plasmid was transformed into N315 and N315Δ sarA , respectively. The LacZ activity was detected and represented as mean ± S.D. ( n = 3). ns indicated no significance, *** P

    Journal: bioRxiv

    Article Title: β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

    doi: 10.1101/353227

    Figure Lengend Snippet: SarA bound to the tlpp cluster promoter region to control the β-lactam-stimulated Tlpp expression in MRSA. (A) β-galactosidase assay. The pOS1- tlpps P reporter plasmid was transformed into N315 and N315Δ sarA , respectively. The LacZ activity was detected and represented as mean ± S.D. ( n = 3). ns indicated no significance, *** P

    Article Snippet: Preparation of the total bacteria proteins and the culture supernatant proteinsOvernight MRSA culture was diluted 1:100 in BHI medium with or without the addition of β-lactam antibiotics, cultivated at 37 °C to an optical density (OD) at 600 nm of 2.0.

    Techniques: Expressing, Plasmid Preparation, Transformation Assay, Activity Assay

    β-lactam treatment enhanced the pathogenesis of MRSA in the mouse subcutaneous infection model. (A) Mice were injected subcutaneously with 5×10 7 bacterial cells. Abscess areas were measured daily. (B) Representative abscesses 7 days after infection. (C) Representative histological examinations (H E stain) of the infected mouse skin. IL-6 (D) and TNFα (E) levels in mouse sera determined by ELISA. BALB/c mice were infected by tail vein injection with 1 × 10 7 of N315, OXA-treated N315, and OXA-treated N315Δ tlpps , respectively. The levels of IL-6 and TNFα in mouse sera were determined 6 h post-infection. Number of mice used, n = 3. PBS and OXA served as the controls. ns represented no significance, * P

    Journal: bioRxiv

    Article Title: β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

    doi: 10.1101/353227

    Figure Lengend Snippet: β-lactam treatment enhanced the pathogenesis of MRSA in the mouse subcutaneous infection model. (A) Mice were injected subcutaneously with 5×10 7 bacterial cells. Abscess areas were measured daily. (B) Representative abscesses 7 days after infection. (C) Representative histological examinations (H E stain) of the infected mouse skin. IL-6 (D) and TNFα (E) levels in mouse sera determined by ELISA. BALB/c mice were infected by tail vein injection with 1 × 10 7 of N315, OXA-treated N315, and OXA-treated N315Δ tlpps , respectively. The levels of IL-6 and TNFα in mouse sera were determined 6 h post-infection. Number of mice used, n = 3. PBS and OXA served as the controls. ns represented no significance, * P

    Article Snippet: Preparation of the total bacteria proteins and the culture supernatant proteinsOvernight MRSA culture was diluted 1:100 in BHI medium with or without the addition of β-lactam antibiotics, cultivated at 37 °C to an optical density (OD) at 600 nm of 2.0.

    Techniques: Infection, Mouse Assay, Injection, H&E Stain, Enzyme-linked Immunosorbent Assay

    Tlpps upregulated in MRSA post-treated with the subinhibitory concentrations of antibiotics. (A) The proteins of different antibiotic-induced MRSA N315 were separated through SDS-PAGE and stained with Coomassie brilliant blue. An untreated N315 served as the negative control, and the molecular weights of the protein marker were indicated on the left. The upregulated protein bands upon β-lactam antibiotic treatment were denoted by yellow triangles. (B) Other major clinically prevalent MRSA strains represented by sequence type (ST) were cultured in the absence or presence of OXA (2 μg/ml, unless specifically stated, S2 Table and S9 Fig). SDS-PAGE was then performed. The yellow triangles indicated that certain upregulated protein bands were similar to those of β-lactam-induced MRSA N315 (ST5). (C) SDS-PAGE analysis of the recombinant Tlpp1 proteins. (D) Western blot analysis of β-lactam-induced proteins in N315. The full-length blot was presented in S10 Fig.

    Journal: bioRxiv

    Article Title: β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

    doi: 10.1101/353227

    Figure Lengend Snippet: Tlpps upregulated in MRSA post-treated with the subinhibitory concentrations of antibiotics. (A) The proteins of different antibiotic-induced MRSA N315 were separated through SDS-PAGE and stained with Coomassie brilliant blue. An untreated N315 served as the negative control, and the molecular weights of the protein marker were indicated on the left. The upregulated protein bands upon β-lactam antibiotic treatment were denoted by yellow triangles. (B) Other major clinically prevalent MRSA strains represented by sequence type (ST) were cultured in the absence or presence of OXA (2 μg/ml, unless specifically stated, S2 Table and S9 Fig). SDS-PAGE was then performed. The yellow triangles indicated that certain upregulated protein bands were similar to those of β-lactam-induced MRSA N315 (ST5). (C) SDS-PAGE analysis of the recombinant Tlpp1 proteins. (D) Western blot analysis of β-lactam-induced proteins in N315. The full-length blot was presented in S10 Fig.

    Article Snippet: Preparation of the total bacteria proteins and the culture supernatant proteinsOvernight MRSA culture was diluted 1:100 in BHI medium with or without the addition of β-lactam antibiotics, cultivated at 37 °C to an optical density (OD) at 600 nm of 2.0.

    Techniques: SDS Page, Staining, Negative Control, Marker, Sequencing, Cell Culture, Recombinant, Western Blot

    Reporter assay for detection of AmpG permease activity. The β-lactamase assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.

    Journal: Frontiers in Microbiology

    Article Title: Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia

    doi: 10.3389/fmicb.2017.00648

    Figure Lengend Snippet: Reporter assay for detection of AmpG permease activity. The β-lactamase assay was performed on Escherichia coli strains BW25113 (parental strain), TP73 (Δ ampD )/pNU305, AR74 (Δ ampG/ Δ ampD )/pNU305/ pACY-AR1 (empty vector), and AR74/pNU305/pAC-Tanf_08635. Activity was determined by chromogenic nitrocefin substrate induced at 0, 30, 60 min and absorbance was measured at OD 486 . Data are representative of three independent experiments with similar results. Each value represents the mean (±SD) of three values measured in one representative assay.

    Article Snippet: Lactamase activity of each strain lysate was assayed using the chromogenic nitrocefin substrate (Calbiochem).

    Techniques: Reporter Assay, Activity Assay, Beta Lactamase Assay, Plasmid Preparation