Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A) ArcA phosphorylation. V. cholerae overexpressing N-terminally His-tagged ArcA WT or ArcA D54A were grown aerobically (O 2 + ), microaerobically (O 2 − ), or microaerobically in the presence of 50 μM CHP. Whole-cell lysates were analyzed by Phos-Tag gel electrophoresis followed by anti-His6 western blotting to visualize ArcA phosphorylation. (B) ROS effects on ArcA repression of sdhC transcription. Wild type, Δ arcA , and arcA C173S mutants containing P sdhc -lacZ reporter plasmids were grown in AKI medium to mid-log phase and treated with or without 60 μM CHP for 1 h. β-Galactosidase activity was measured and normalized against viable cell numbers (SU, special units = β-gal unit/CFU × 10 6 ). The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ****, p < 0.0001 (Student’s t test); ns, no significance. (C) EMSA assays. Purified ArcA wild type and ArcA-mutant derivative proteins were treated with DTT (R, reduced), carbamoyl phosphate (P, phosphorylated), and CHP (O, oxidized) before incubating with the 32 P-labeled sdhC promoter DNA. The reaction mixes were separated on a native gel and imaged on a Typhoon phosphorimager.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Phospho-proteomics, Nucleic Acid Electrophoresis, Western Blot, Activity Assay, Standard Deviation, Purification, Mutagenesis, Labeling

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A and B) ArcA-ArcA interaction. E. coli BTH101 containing pKT25- arcA and pUC18c- arcA and derivatives were grown for 12 h at 37°C in LB aerobically or microaerobically (A) or microaerobically with or without 50 μM CHP (B). Single-step β-galactosidase activity assays were performed, and the LacZ unit was calculated by the formula described in . The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ***, p < 0.001 (Student’s t test); ns, no significance. (C) V. cholerae containing overexpressed His6-tagged ArcA variants were grown under the virulence-inducing condition. When indicated, 50 μM CHP was added during ArcA induction. Cell lysates (normalized by optical density at 600 nm [OD 600 ]) with or without 100 mM BME were separated on a non-reducing 12% SDS-polyacrylamide gel followed by western blotting analysis using anti-His6 antibody; O-M, oxidized monomer; R-M, reduced monomer; O-D, oxidized dimer. (D) Full MS spectrum of the [M + 3H] 3+ ( m/z = 675.30) ions of the disulfide-linked ALLHFC 173 ENPGK and FC 233 GDLED. (E) Working model. ArcA in the reduced unphosphorylated state is in an inactive form as monomers. A microaerobic environment leads ArcA phosphorylation and thus ArcA-ArcA interaction, allowing target DNA binding. When cells are challenged by ROS, ArcA phosphorylation is abolished, but an intramolecular disulfide bond is formed, introducing a conformational change that retains ArcA-ArcA interaction and DNA binding.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Activity Assay, Standard Deviation, Western Blot, Phospho-proteomics, Binding Assay

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, cDNA Synthesis, Control, Software, Sequencing