algr d54e xbai r  (New England Biolabs)


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    Structured Review

    New England Biolabs algr d54e xbai r
    Phosphorylated <t>AlgR</t> and AlgR <t>D54E</t> bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.
    Algr D54e Xbai R, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility"

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00726-13

    Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.
    Figure Legend Snippet: Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Techniques Used: In Vitro, Binding Assay, Purification, Incubation

    AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or
    Figure Legend Snippet: AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Techniques Used: Thin Layer Chromatography, Colorimetric Assay

    Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P
    Figure Legend Snippet: Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Techniques Used: Negative Control

    AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P
    Figure Legend Snippet: AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Techniques Used: Activity Assay

    Related Articles

    Clone Assay:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: All cloning techniques were performed using E. coli DH5α to propagate and maintain plasmids, unless otherwise indicated. .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Amplification:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: To make the algR complementation vector, the algR coding region was PCR amplified from the PAO1 genome using primers algR-CdgRgn-KpnI-F and algR-CdgRgn-HindIII-R. .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Mutagenesis:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs). .. To construct the pHERD30T rhlAB vector, the rhlAB genes were amplified from the PAO1 chromosome with primers rhlAB EcoRI F and rhlAB HindIII R and were subcloned into the pHERD30T vector using the EcoRI and HindIII restriction sites.

    Construct:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs). .. To construct the pHERD30T rhlAB vector, the rhlAB genes were amplified from the PAO1 chromosome with primers rhlAB EcoRI F and rhlAB HindIII R and were subcloned into the pHERD30T vector using the EcoRI and HindIII restriction sites.

    Generated:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: The pHERD30T- algR D54N vector was generated in a similar manner using chromosomal DNA extracted from WFPA8 ( ). .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Polymerase Chain Reaction:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: This PCR product was digested with KpnI and HindIII and was ligated into pHERD30T ( ) to generate pHERD30T- algR . .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Plasmid Preparation:

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility
    Article Snippet: The pHERD30T- algR D54N vector was generated in a similar manner using chromosomal DNA extracted from WFPA8 ( ). .. To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

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    New England Biolabs algr d54e xbai r
    Phosphorylated <t>AlgR</t> and AlgR <t>D54E</t> bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.
    Algr D54e Xbai R, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/algr d54e xbai r/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    algr d54e xbai r - by Bioz Stars, 2020-04
    86/100 stars
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    Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Phosphorylated AlgR and AlgR D54E bound to the fimU promoter. The effect of in vitro phosphorylation of AlgR on binding to an 82-bp fimU DNA fragment was compared with the binding of purified AlgR D54N or AlgR D54E using EMSA. (A) Diagram of the 82-bp fragment used in EMSAs and its location within the fimT-fimU intergenic region. (B) Increasing amounts of the phosphate donor, AcP, were added to the AlgR protein in order to determine the effect of phosphorylation on DNA binding. Wild-type AlgR protein was pretreated with AcP for 30 min prior to binding reactions. Lanes 1 to 8, fimU DNA alone (lane 1) or in the presence of AlgR without AcP (lane 2) or with increasing concentrations of AcP (lanes 3 to 8). Lanes 9 to 12, the AlgR-P–DNA complex was competed with the addition of nonradiolabeled fimU DNA. (C) Phosphorylated AlgR protein was compared with untreated AlgR for DNA binding. Purified AlgR protein was pretreated either with water only or with AcP (500× molar excess of protein) prior to binding reactions. Lanes 1 to 10, fimU DNA incubated with a no-protein control (lane 1), increasing concentrations of AlgR (lanes 2 to 5) or AlgR-P (lanes 6 to 9), or 5 mM AcP only (lane 10). Lanes 11 to 13, 5 nM pscEF DNA incubated with a no-protein control (lane 11), 2.5 μM AlgR (lane 12), or 2.5 μM AlgR-P (lane 13). (D) Purified AlgR D54N or AlgR D54E protein was tested for binding to fimU DNA. Lane 1, no-protein control; lanes 2 to 5, increasing concentrations of AlgR D54N; lanes 6 to 9, increasing concentrations of AlgR D54E (lanes 6 to 9). Open arrowheads, unbound DNA; filled arrowheads, AlgR-DNA complexes. All concentrations given in the figure are final binding reaction concentrations.

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: In Vitro, Binding Assay, Purification, Incubation

    AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR phosphorylation was required for maximal rhamnolipid production. (A) Thin-layer chromatography. Strains PAO1 (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAO1 Δ rhlA (Δ rhlA ) were grown for 16 h in TSB plus 1% glycerol. Mono, monorhamnolipid band; Di, dirhamnolipid band. (B) Strains for which results are shown in panel A were tested for rhamnolipid production by use of the orcinol colorimetric assay and comparison with a standard curve (see Materials and Methods). Values were normalized to total-protein values. (C) The extent of hemolysis due to rhamnolipid production was measured. Cultures were grown for 24 h in a phosphate-limiting medium (tryptic soy broth supplemented with 1% glucose), and cell-free supernatants were heat treated to denature polypeptide-based hemolysins. Serial dilutions were applied to 1% horse red blood cells for 1 h at 37°C, and the percentage of hemolysis was calculated based on total and no hemolysis. Statistical analysis was performed by one-way analysis of variance. n.s., not significant. Asterisks indicate P values of 0.05 to 0.01 (*), 0.01 to 0.001 (**), or

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Thin Layer Chromatography, Colorimetric Assay

    Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: Effects of different algR alleles on twitching and swarming motility. (A) Subsurface twitching motilities were analyzed at 48 h after inoculation in LB–1% agar, and the average diameters of twitching zones were measured. Strains PAO1 ( algR + ) (WT), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54 E) were analyzed. PAO1A ( pilA :: Tc ) was used as a negative control. Statistical analysis was performed by one-way analysis of variance. n.s., not significant; ***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Negative Control

    AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Journal: Journal of Bacteriology

    Article Title: Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

    doi: 10.1128/JB.00726-13

    Figure Lengend Snippet: AlgR D54E increased fimU transcription. (A) Diagram showing the genotypes of the following strains: PAO1 ( algR + ), PSL317 (Δ algR ), WFPA8 ( algR D54N), PAO1 algR D54E, PAZ (Δ algZ ), and PAZ algR D54E (Δ algZ algR D54E). The dashed regions represent the deletion of the gene indicated. (B) The strains diagramed in panel A were tested for fimU transcriptional activity. Strains contained a fimU :: lacZ transcriptional reporter in the chromosomal attB locus. β-Galactosidase activities from 21-h LB cultures grown at 37°C were assayed. Significant differences from the wild type by Student t tests are indicated (***, P

    Article Snippet: To create pHERD30T- algR D54E, the codon encoding Asp 54 (GAT) in pHERD30T- algR was mutated to Glu (GAA) using primers algR-D54E-XbaI-F and algR-D54E-XbaI-R with a Phusion site-directed mutagenesis kit (New England BioLabs).

    Techniques: Activity Assay