e coli rna polymerase holoenzyme  (New England Biolabs)

Figure Legend Snippet: Constitutive expression of rpfG restores motility and EPS production of the mutant Δ tfmR . And Xoc TfmR binds directly to the promoter of RpfG and activates its transcription. ( A ) Constitutive expression of rpfG restores motility of the mutant Δ tfmR . ( i ) Example photo of a bacterial strain. ( ii ) Mean measurements of colony diameter for each strain on “swarming” plates. ( iii ) Mean measurements of colony diameter for each strain on “swimming” plates. Data shown are the mean ± SD ( n = 10). Significance was determined by ANOVA and Dunnett’s post hoc test for comparison with to the wild type. ** p < 0.01; n.s., not significant. ( B ) Constitutive expression of rpfG restores the yield of the mutant Δ tfmR EPS. ( i ) Xoc strains were grown on NA plates supplemented with 2% sucrose for 3 days. ( ii ) Xoc strains were cultured in NB medium supplemented with 2% sucrose for 3 days and EPS was precipitated from the culture supernatant. Values given are the means ± SD of triplicate measurements from a representative experiment, and significance was determined by analysis of variance (ANOVA) and Dunnett’s post hoc test for comparison with the wild type. * p < 0.05; n.s., not significant. Similar results were obtained in two other independent experiments. ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of rpfG ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) ß–Glucuronidase (GUS) activity of the gusA reporter of the rpfG gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) Detection of Δ tfmR mutant and wild–type expression of rpf genes in NB medium ( i ), or XOM2 medium ( ii ), revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( F ) Fold enrichment of the promoter region of rpfG in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t -test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( G ) In vitro transcription experiments showing TfmR activates the transcription of rpfG . RNA was produced from a 323 bp template DNA fragment containing the rpfG promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 150 bp template DNA fragment of the rpfG coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Techniques Used: Expressing, Mutagenesis, Comparison, Cell Culture, Electrophoretic Mobility Shift Assay, Negative Control, Activity Assay, Standard Deviation, Quantitative RT-PCR, In Vitro, Produced, Sequencing

Figure Legend Snippet: Xoc TfmR binds directly to the promoter of HrpX and activates its transcription. ( A ) ß–Glucuronidase (GUS) activity of the gusA reporter of the hrpG and hrpX gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( B ) Detection of Δ tfmR –mutant and wild-type expression of T3SS genes in NB medium ( i ), or XOM2 medium ( ii ) revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of hrpX ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) Fold enrichment of the promoter region of hrpX in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t –test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) In vitro transcription experiments showing TfmR activates the transcription of hrpX . RNA was produced from a 371 bp template DNA fragment containing the hrpX promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 161 bp template DNA fragment of the hrpX coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Techniques Used: Activity Assay, Mutagenesis, Standard Deviation, Expressing, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Negative Control, In Vitro, Produced, Sequencing