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. 2017 May 26;6:e25624. doi: 10.7554/eLife.25624

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© 2017, Mishra et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 8. — Oxidized (WhiB4-SS) and reduced (WhiB4-SH) forms of apo-WhiB4 were prepared. The concentrations of apo-WhiB4 used for EMSAs were 0.5, 1, 2, and 4 μM. EMSA reactions were performed with 0.5 nM ³²P-labelled blaC (A), blaR (B) and whiB4 (C) promoter DNA fragments. C: DNA binding in the absence of WhiB4 in each panel. (D) Effect of WhiB4 on in vitro transcription. Single-round transcription assays show that RNAP-σ^A efficiently directs transcription from the blaC promoter. 100 nM of blaC promoter DNA fragment was pre-incubated with either 1 μM WhiB4-SS or WhiB4-SH and subjected to transcription by RNAP-σ^A as described in Materials and methods. C: blaC transcript in the absence of WhiB4. (E) 100 μg of cell-free lysates derived from exponentially grown (OD₆₀₀ of 0.6) wt Mtb, MtbΔwhiB4 and whiB4-OE were used to hydrolyze nitrocefin. β-lactamase activity was measured by monitoring absorbance of hydrolyzed nitrocefin at 486 nm as described in Materials and methods. The fold change ratios clearly indicate a significantly higher or lower β-lactamase activity in MtbΔwhiB4 or whiB4-OE, respectively, as compared to wt Mtb. p-Values are shown for each comparison. (F) whiB4-OE strain was pre-treated with 5 mM of DTT or Diamide and β-lactamase activity in cell-free lysates was compared to MtbΔwhiB4 over time. *p≤0.05, **p≤0.01 and ***p≤0.001. Data are representative of at least two independent experiments done in duplicate.

DOI: http://dx.doi.org/10.7554/eLife.25624.023

Figure 8—figure supplement 1. — Oxidized (WhiB4-SS) and reduced (WhiB4-SH) forms of apo-WhiB4 were prepared. The concentrations of apo-WhiB4 used for EMSAs were 0.5, 1, 2, and 4 μM. EMSA reactions were performed with 0.5 nM ³²P-labelled blaC (A), blaR (B) and whiB4 (C) promoter DNA fragments. C: DNA binding in the absence of WhiB4 in each panel. (D) Effect of WhiB4 on in vitro transcription. Single-round transcription assays show that RNAP-σ^A efficiently directs transcription from the blaC promoter. 100 nM of blaC promoter DNA fragment was pre-incubated with either 1 μM WhiB4-SS or WhiB4-SH and subjected to transcription by RNAP-σ^A as described in Materials and methods. C: blaC transcript in the absence of WhiB4. (E) 100 μg of cell-free lysates derived from exponentially grown (OD₆₀₀ of 0.6) wt Mtb, MtbΔwhiB4 and whiB4-OE were used to hydrolyze nitrocefin. β-lactamase activity was measured by monitoring absorbance of hydrolyzed nitrocefin at 486 nm as described in Materials and methods. The fold change ratios clearly indicate a significantly higher or lower β-lactamase activity in MtbΔwhiB4 or whiB4-OE, respectively, as compared to wt Mtb. p-Values are shown for each comparison. (F) whiB4-OE strain was pre-treated with 5 mM of DTT or Diamide and β-lactamase activity in cell-free lysates was compared to MtbΔwhiB4 over time. *p≤0.05, **p≤0.01 and ***p≤0.001. Data are representative of at least two independent experiments done in duplicate.

DOI: http://dx.doi.org/10.7554/eLife.25624.023