The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV)

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7’s ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the −35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7–SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.


Construction of 10xHis:WhiB7 expression vector mutants
The QuickChange Lightning mutagenesis kit was used to mutate pETB7sm to change WhiB7's EPW sequence (aa 63-65) immediately upstream of the GVWGG turn. The sequence was mutated to the WhiB3 like EPY using the primers B7_EPY_F and B7_EPY_R to construct pETB7epy, and to the WhiB3 like VEY using the primers B7_VEY_F and B7_VEY_R to construct pETB7vey. The glutamate was also mutated to an aspartate using the primers B7_D_F and B7_D_R to construct pETB7d. The fourth cysteine (latter of the CXXC motif) was mutated to an alanine using the primers B7_48_F and B7_48_R to construct pETB748. Lastly, the CXXC motif cysteines were mutated to alanines using primers B7_4548_F and B7_4548_R to construct pETB74548.

Construction of WhiB7 and SigA co-expression vector
A C-terminal fragment of M. smegmatis sigA similar to that used to show WhiB3-SigA interaction (1), including regions 2.5, 3.1, 3.2, 4.1 and 4.2 (2), was amplified by PCR from pTRG170 (described below) using the primers CD_R42SM_F and CD_R42SM_R. This added a N-terminal strepII tag. The product was digested with NdeI/ KpnI, and cloned into similarly digested pColaDuet-1 to construct pSigA. 10xHis-tagged whiB7 was extracted from pETB7sm using NcoI/ BamHI and cloned into similarly digested pSigA to construct the 10xHis-WhiB7 and strepII-SigA co-expression vector pSigAB7.

Construction of co-expression vector mutants
Mutant forms of 10xHis-WhiB7 were extracted by NcoI/ BamHI from pETB748, and pETB74548 and cloned into similarly digested pSigAB7 to construct pSigAB748 and pSigAB74548, respectively.

Construction of WhiB7 and region 4 of SigA co-expression vector
A similar alignment to that used by Vassylyev et al. (2) was used to identify the discreet regions of M. smegmatis SigA. The primers CD_R42short_F and CD_R42SM_R were used to PCR amplify region 4 of SigA. The product was then digested with NdeI/ KpnI and cloned into similarly digested pSigAB7 to construct the 10xHis-WhiB7 and strepII-SigA(region 4) coexpression vector pR4B7.

Construction of Bait constructs
An AT-hookless whiB7 was amplified by PCR from the M. smegmatis genome using the forward primer B7smF and the reverse primer B7smR_AT. The PCR product was digested with EcoRI and ligated into similarly digested pBT to construct pBTW7C19. A 26 amino acid fragment of WhiB7, spanning the region downstream of the last cysteine and upstream of the AT-hook, was ordered from IDT (www.idtdna.com); B7midFrag_F and B7midFrag_R. The oligonucleotides were dissolved to 10 pmol/ L and mixed at equal volumes. The mixture was heated to 95 ºC for 5 minutes and left to cool to room temperature. The resulting dimers were ligated into EcoRI/ XhoI digested pBT to construct pBTW7mid.

Construction of pBTW7C19 bait mutants
The QuickChange Lightning mutagenesis kit was used to mutate pBTW7C19 to change WhiB7's EPW sequence (aa 63-64) immediately upstream of the GVWGG turn. The sequence was mutated to the WhiB3 like EPY using the primers B7_EPY_F and B7_EPY_R to construct pBTW7epy, and to the WhiB3 like VEY using the primers B7_VEY_F and B7_VEY_R to construct pBTW7vey. The glutamate was also mutated to an aspartate using the primers B7_D_F and B7_D_R to construct pBTW7d. A glutamate further downstream (E71) was also mutated using the primers B7_71D_F and B7_71D_R to construct pBTW71d. The fourth cysteine (latter of the CXXC motif) was mutated to an alanine using the primers B7_48_F and B7_48_R to construct pBTW748. Lastly, the CXXC motif cysteines were mutated to alanines using primers B7_4548_F and B7_4548_R to construct pBTW74548.

Construction of pTRG170
sigA from M. smegmatis (MSMEG_2758) was amplified by PCR using the primers SigAsmF and SigAsmR. The PCR product was digested with EcoRI/ SpeI, and ligated into similarly digested pTRG to construct pSigASM. pSigASM was digested with EcoRI/ NcoI, and self-ligated. Sequencing confirmed that this resulted in a 901 bp deleted region spanning from immediately downstream of the pTRG BamHI site to the ATG within the NcoI site in sigA leading to pTRG170. This expressed a C-terminal fragment of M. smegmatis  which is 100% similar (99.6% identical) to that of M. tuberculosis SigA (aa 359-528). Previous studies investigating WhiB3-SigA interaction used aa 369-528 as the SigA target (1).

Construction of a pTRG170 R515H mutant
The QuickChange Lightning mutagenesis kit was used to mutate pTRG170 with the primers mutR42_F and mutR42_R to construct pTRG170.515. This resulted in a MSMEG_2758 G1358A mutation leading to expression of SigA Arg453His. This corresponds to the Mtb SigA R515H mutation. The Mtb numbering will be used to simplify discussion of previous WhiB3-SigA results.

Construction of M. smegmatis Sig515
The R515H mutated region 4.2 of sigA was removed from pTRG170.515 using NcoI/ HindIII. It was combined with the XhoI/ NcoI sigA fragment from pSigASM and cloned into XhoI/ HindII digested pYUB854 to construct pSig515. The downstream region of sigA was then amplified by PCR from the M. smegmatis genome using the primers SigAflank_F and SigAflank_R. The PCR product was digested with XbaI/ KpnI and cloned into similarly digested pSig515 to construct pSig515KI. This reversed the orientation of the hygromycin resistance gene so that it matched the orientation of sigA. pSig515KI was then digested with XhoI/ KpnI and the digest was used to construct M. smegmatis Sig515 using mycobacterial recombineering (3) ( Figure 5A). Three randomly picked recombinants were used for all determinations. sigA from the mutants was amplified using the primers SigAsmF and SigAsmR. The product was cloned, utilizing the 5' addition of A overhangs, into pGEM-T easy and sequenced to assure the R515H mutation. The presence of whiB7 in these strains was confirmed by PCR using the primers 497_F and B7smR.
A 10 mL syringe was used as a column containing 1 mL of Ni-NTA resin (Qiagen) held at 4 ºC. The column was washed with 6 column volumes (CV) of wash buffer (50 mM Na 2 PO 4 300 mM NaCl, 10 mM imidazole, pH 8) and the flow rate adjusted to 1 mL/ min. The filtered supernatant was then passed over the column resulting in the column turning brown. The column was then washed with six 10 CV loads of wash buffer containing an increasing amount of imidazole (50, 60, 70, 80, 90, and 100 mM). Finally elution buffer (50 mM Na 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8) was applied to the column. This resulted in a clear, brown eluate. Fractions which were visibly dark brown were pooled, DTT was added to a final concentration of 2 mM, and aliquots were immediately frozen in liquid nitrogen and stored at -80 ºC until use. Table S1. Oligonucleotides