Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

Additional file 1:

Figure 1 – Characterization of WhiB1 as a bait. (A) Repression assay to asses the expression and binding of 'bait' to the LexA operator. Four independent transformants of each of the clones were assayed for β-gal expression. Saccharomyces cerevisiae cells (~2 × 10⁷) containing constructs (labeled on the x-axis), were permeabilized with 3 drops of chloroform and 2 drops of 0.1% SDS by vigorous vortexing. Reaction was initiated by adding 0.2 ml ONPG (4 mg/ml) in 'Z buffer' for 3.5 minutes at 28°C. The β-gal activity is expressed as units, on y-axis. (B) WhiB1 lacks intrinsic transcription activity. Saccharomyces cerevisiae cells were transformed with pDBD-B1 (bait), pSH18-34 which contains β-gal as a reporter gene and library plasmid pJG4-5. The β-gal expression was monitored on the Gal⁺Raff⁺Ura^-X-gal plate. The panels indicate S. cerevisiae containing (1) pSH18-34 + pJG4-5 + pDBD-B1, test (2) pSH18-34 + pJG4-5+ pSH17-4, positive control (3) pSH18-34 + pJG4-5 + pRFHM1, negative control. Figure 2 – Homology model of Mtb GlgB. Three domains are colored differently: N2 domain-orange; TIM-barrel domain-magenta; C-terminal domain-blue. The linker regions between the domains are shown in red. The position of the cysteine residues (C¹⁹³, C⁶¹⁷and C⁶⁵⁸) on the individual domains is shown as green sticks encircled in dotted boundaries. Figure 3 – Over-expression and purification of TrxB/Rv1471 (1) and TrxC/3914 (2) of Mtb. The proteins were overexpressed in E. coli BL21 (DE3) and were purified by Ni²⁺-NTA affinity chromatography. Lanes are indicated on the top.

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Additional file 2:

Table 1 – Protein disulfide reductase activity of Mtb TrxB and TrxC. Table 2 – List of primers used in this study

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