Journal: Frontiers in Microbiology

Article Title: SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

doi: 10.3389/fmicb.2020.01856

Figure Lengend Snippet: Strains.

Article Snippet: 20 nM PCR fragments were incubated with purified SlyA and ppGpp (TriLink Biotechnologies) (see legends of and for the concentrations), in a 20 μl final reaction buffer containing 25 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM EDTA, 50 mM KCl, and 5% glycerol.

Techniques: Transduction

Journal: Frontiers in Microbiology

Article Title: SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

doi: 10.3389/fmicb.2020.01856

Figure Lengend Snippet: Effect of ppGpp on SlyA binding to DNA. Electrophoretic mobility shift assays were performed using purified SlyA from E. coli [at 50 (+) or 100 (++) nM] or purified SlyA from Salmonella for the pagC experiment [at 200 (+) or 400 (++) nM]. For each SlyA concentration, 0, 50, or 100 μM ppGpp were added.

Article Snippet: 20 nM PCR fragments were incubated with purified SlyA and ppGpp (TriLink Biotechnologies) (see legends of and for the concentrations), in a 20 μl final reaction buffer containing 25 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM EDTA, 50 mM KCl, and 5% glycerol.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Concentration Assay

Journal: Frontiers in Microbiology

Article Title: SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

doi: 10.3389/fmicb.2020.01856

Figure Lengend Snippet: In vivo induction by SlyA does not require ppGpp. (A) Comparison of slyA transcriptional fusion activity in wild type MG1655, Δ slyA (EB1073), ppGpp° (EB425), and Δ slyA_ ppGpp° (EB1077) strains grown overnight at 30°C in LB. (B) Effect of pBAD-SlyA overproduction in Δ slyA or Δ slyA_ ppGpp° strains on the expression of slyA , paaA , pagC _Stm, and hlyE transcriptional fusions, in the same conditions as in . The activities correspond to the ratio between GFP fluorescence and OD 600nm of six (A) or four (B) replicates, given in arbitrary units (A.U.). The error bars show the SEM.

Article Snippet: 20 nM PCR fragments were incubated with purified SlyA and ppGpp (TriLink Biotechnologies) (see legends of and for the concentrations), in a 20 μl final reaction buffer containing 25 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM EDTA, 50 mM KCl, and 5% glycerol.

Techniques: In Vivo, Comparison, Activity Assay, Expressing, Fluorescence

Journal: Frontiers in Microbiology

Article Title: SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

doi: 10.3389/fmicb.2020.01856

Figure Lengend Snippet: hlyE activation by SlyA in global regulatory mutants. Induction of the hlyE transcriptional fusion by pBAD-slyA_ecoli was tested in the same conditions as in , in the indicated mutant strains: wt (MG1655), Δ slyA (EB1073), Δ slyA_ ppGpp (EB1077), Δ slyA Δ dksA (EB1100), Δ cyaA (EB781), Δ fis (EB743), and Δ hns (EB951). The activities correspond to the ratio between GFP fluorescence and OD 600nm of 4 (A,C) or 3 (B) replicates, given in arbitrary units (A.U.). The error bars show the SEM.

Article Snippet: 20 nM PCR fragments were incubated with purified SlyA and ppGpp (TriLink Biotechnologies) (see legends of and for the concentrations), in a 20 μl final reaction buffer containing 25 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM EDTA, 50 mM KCl, and 5% glycerol.

Techniques: Activation Assay, Mutagenesis, Fluorescence

Journal: Frontiers in Microbiology

Article Title: SlyA Transcriptional Regulator Is Not Directly Affected by ppGpp Levels

doi: 10.3389/fmicb.2020.01856

Figure Lengend Snippet: Effect of ppGpp on SlyA dimerization in vivo . (A) MG1655_SlyA-3Flag (EB1106) and ppGpp°_SlyA-3Flag (EB1110) strains were grown to OD600 = 1.3. (B) Strain MG1655_SlyA-3Flag (EB1106) was transformed by plasmids pALS10, pALS13, and pALS14 (pEB0697, pEB0698, and pEB0699, respectively) . These transformed strains were grown to OD600 = 1.5 and then relA expression was induced with 1 mM IPTG for 30 min. Then, for both panels, the cells were cross-linked with formaldehyde as described in section “Materials and Methods.” +F, with formaldehyde; –F, without formaldehyde; +/H, with formaldehyde and then heated at 96°C. After SDS-PAGE and Western blot, the SlyA-3Flag tag was detected with monoclonal anti-Flag M2.

Article Snippet: 20 nM PCR fragments were incubated with purified SlyA and ppGpp (TriLink Biotechnologies) (see legends of and for the concentrations), in a 20 μl final reaction buffer containing 25 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1 mM CaCl 2 , 0.5 mM EDTA, 50 mM KCl, and 5% glycerol.

Techniques: In Vivo, Transformation Assay, Expressing, SDS Page, Western Blot