Journal: Nature Microbiology

Article Title: KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid

doi: 10.1038/s41564-024-01915-3

Figure Lengend Snippet: a , Promoter scaffold from the RK2 korABF operon ( PkorA ) is shown with core promoter elements (underlined), OA (magenta box), and OB (green box). Differences to the PkorA :λ P R discriminator are depicted. b , Deconvolved nMS spectra of E. coli Eσ 70 holoenzyme assembled on 100 bp PkorA DNA (Eσ 70 :DNA) with and without 2.5-fold excess KorA dimer in 150 mM ammonium acetate pH 7.5 and 0.01% Tween-20. c , Deconvolved nMS spectra of Eσ 70 :DNA with and without 2.5-fold excess KorB dimer in 500 mM ammonium acetate pH 7.5 and 0.01% Tween-20. d , Top: representative gel close-up on the abortive RNA product (5′-ApUpG-3′) transcribed by Eσ 70 in in vitro abortive initiation half-life assays on the two PkorA linear scaffold variants. Bottom: plot of fraction of competitor-resistant open complexes (from normalized abortive RNA band intensities) against time. Data points from three experimental replicates are mean values ± s.e.m. with an exponential trend line fit. Some error bars are too small or lead to negative values and thus were omitted. Estimated half-lives are shown adjacent to exponential decay trend lines. e , WT PkorA and PkorA :λ P R discriminator in vitro transcription repression of Eσ 70 in the presence of fivefold excess KorA and/or KorB. Data points from at least three experimental replicates are normalized to holoenzyme only control as mean values ± s.e.m. Eσ 70 + KorA and Eσ 70 + KorA + KorB conditions on WT PkorA has n = 4, while the rest are n = 3; this remains valid as repression quantities are normalized to a Eσ 70 -only control and background-corrected for each gel run, and s.e.m. are considered in statistical analysis. P values were calculated by unpaired two-tailed Welch’s t -tests; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. The P values are 3.66 × 10 −3 (Eσ 70 + KorA), 0.635 × 10 −3 (Eσ 70 + KorB) and 31.5 × 10 −3 (Eσ 70 + KorA + KorB).

Article Snippet: C-terminally His-tagged KorA and KorB (WT and mutants) were expressed from the plasmid pET21b in E. coli Rosetta (BL21 DE3) pLysS competent cells (Merck).

Techniques: In Vitro, Abortive Initiation Assay, Control, Two Tailed Test

Journal: Nature Microbiology

Article Title: KorB switching from DNA-sliding clamp to repressor mediates long-range gene silencing in a multi-drug resistance plasmid

doi: 10.1038/s41564-024-01915-3

Figure Lengend Snippet: a , Representative urea-PAGE gel closeup on the abortive RNA product (5’-ApUpG*-3’; *radiolabelled on guanosine alpha-phosphate) transcribed in in vitro abortive initiation assays on WT PkorA and the PkorA :λ P R discriminator mutant linear DNA scaffolds with different mixes of Eσ 70 and/or fivefold excess KorA, fivefold excess KorB and/or saturating CTP. b , WT PkorA in vitro transcription repression of E. coli RNAP:σ70 holoenzyme (Eco Eσ70) in the presence of fivefold excess KorA and/or KorB. Experiments were repeated at least three times. All values are normalized to holoenzyme only control as mean values ± SEM. Eσ 70 + KorB condition has n = 3 while the rest are n = 4, and this remains valid as repression quantities are normalized to a Eσ 70 -only control and background-corrected for each gel run, and SEMs are considered in statistical analysis. P value was calculated by an unpaired Welch’s t-test; * p ≤ 0.05, ** p ≤ 0.01. The P values are 0.1827 (KorA vs KorA + KorB), 0.0281 (KorA vs KorB), and 0.0067 (KorB vs KorA + KorB). c , Deconvolved native mass spectra of Eσ 70 (5 μM) with 2.5-fold excess KorA dimer electrospray ionized in buffer of 300 mM or 150 mM ammonium acetate pH 8.0 and 0.01% Tween-20. No peak of Eσ 70 :KorA was observed.

Article Snippet: C-terminally His-tagged KorA and KorB (WT and mutants) were expressed from the plasmid pET21b in E. coli Rosetta (BL21 DE3) pLysS competent cells (Merck).

Techniques: In Vitro, Abortive Initiation Assay, Mutagenesis, Control