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. 2011 Mar;39(6):2210-20.
doi: 10.1093/nar/gkq1149. Epub 2010 Nov 12.

Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase

Jee-Hyun Kim  1 , Jun-Rong Wei, Joshua B Wallach, Rebekkah S Robbins, Eric J Rubin, Dirk Schnappinger
Affiliations

Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase

Jee-Hyun Kim et al. Nucleic Acids Res. 2011 Mar.

Abstract

Using a component of the Escherichia coli protein degradation machinery, we have established a system to regulate protein stability in mycobacteria. A protein tag derived from the E. coli SsrA degradation signal did not affect several reporter proteins in wild-type Mycobacterium smegmatis or Mycobacterium tuberculosis. Expression of the adaptor protein SspB, which recognizes this modified tag and helps deliver tagged proteins to the protease ClpXP, strongly decreased the activities and protein levels of different reporters. This inactivation did not occur when the function of ClpX was inhibited. Using this system, we constructed a conditional M. smegmatis knockdown mutant in which addition of anhydrotetracycline (atc) caused depletion of the beta subunit of RNA polymerase, RpoB. The impact of atc on this mutant was dose-dependent. Very low amounts of atc did not prevent growth but increased sensitivity to an antibiotic that inactivates RpoB. Intermediate amounts of RpoB knockdown resulted in bacteriostasis and a more substantial depletion led to a decrease in viability by up to 99%. These studies identify SspB-mediated proteolysis as an efficient approach to conditionally inactivate essential proteins in mycobacteria. They further demonstrate that depletion of RpoB by ∼ 93% is sufficient to cause death of M. smegmatis.

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Figures

Figure 1.
Figure 1.
Effect of SsrA tags and E. coli SspB on GFP, RFP and luciferase (LuxAB) in M. smegmatis. (A) Relative fluorescence units of M. smegmatis gfp, M. smegmatis gfp-ssrAec and M. smegmatis gfp-DAS+4. White bars indicate strains without SspB; hatched bars indicate strains with a constitutively expressed SspB. n/d: not determined. (B) GFP levels detected by immunoblotting in the same strains as in (A). Dihydrolipoamide acyltransferase (DlaT) is used as a loading control. (C) Relative fluorescence units of M. smegmatis rfp, M. smegmatis rfp-ssrAec, M. smegmatis rfp-DAS+4, and six other M. smegmatis strains expressing variously tagged RFP. White bars indicate strains without SspB; hatched bars indicate strains with a constitutively expressed SspB. n/d: not determined. (D) Relative luminescence units of M. smegmatis luxAB, M. smegmatis luxAB-ssrAec and M. smegmatis luxAB-DAS+4. White bars indicate strains without SspB; hatched bars indicate strains with a constitutively expressed SspB. n/d: not determined. (E) Relative luminescence units of M. smegmatis that chromosomally express luxAB, luxAB-ssrAec and luxAB-DAS+4 with or without constitutively expressed SspB. Open bars indicate strains without SspB; hatched bars indicate strains with a constitutively expressed SspB. n/d: not determined. (F) LuxB and SspB levels detected by immunoblotting in the same strains as (D). DlaT is used as a loading control for each immunoblot. Data in (A,C–E) are means ± SD of 10–20 replicates from two or three independent experiments.
Figure 2.
Figure 2.
Inducible degradation of DAS+4-tagged proteins by SspB in M. smegmatis. (A) Relative fluorescence units of M. smegmatis gfp-DAS+4 without sspB, with constitutively expressed sspB, or with tetR and TetR-regulated sspB. White bars indicate strains cultured in the absence of the inducer anhydrotetracycline (atc); hatched bars indicate those cultured in the presence of atc. (B) Relative fluorescence units of M. smegmatis rfp-DAS+4 without sspB, with constitutively expressed sspB, or with tetR and TetR-regulated sspB. Open bars indicate strains cultured in the absence of anhydrotetracycline (atc); hatched bars indicate those cultured in the presence of atc. (C) Relative luminescence units of M. smegmatis luxAB-DAS+4 without sspB, with constitutively expressed sspB, or with tetR and TetR-regulated sspB. White bars indicate strains cultured in the absence of atc; hatched bars indicate those cultured in the presence of atc. (D) Relative luminescence units of M. smegmatis luxAB-DAS+4 with tetR and TetR-regulated sspB cultured with different concentrations of atc. M. smegmatis luxAB-DAS+4 without sspB and M. smegmatis luxAB-DAS+4 with constitutively expressed sspB cultured in the absence of atc serve as controls. Data are means ± SD of 10–20 replicates from two or three independent experiments.
Figure 3.
Figure 3.
Kinetics of GFP-DAS+4 and LuxAB-DAS+4 degradation in M. smegmatis. (A) M. smegmatis gfp-DAS+4, tetR and TetR-regulated sspB were grown to an optical density of 0.2. Each half of the volume was then cultured without (solid line) or with (dashed line) 100 ng/ml atc. Relative fluorescence units were determined at the indicated time points. M. smegmatis gfp-DAS+4 without sspB (closed circles) and with constitutively expressed sspB (open circles) cultured in the absence of atc and measured at 0 and 24 h serve as controls. (B) GFP levels detected by immunoblotting from cultures in (A). A non-specific band recognized by the anti-GFP antibody serves as a loading control for each immunoblot. (C) Relative luminescence units of M. smegmatis luxAB-DAS+4, tetR and TetR-regulated sspB in the absence (solid line) and presence (dashed line) of 100 ng/ml atc. LuxB used in these experiments contained an N-terminal FLAG tag for immunoblotting. (D) LuxB and SspB levels detected by immunoblotting from cultures in (C). DlaT serves as a loading control for each immunoblot. (E) M. smegmatis luxAB-DAS+4 without sspB (left panel), with constitutively expressed sspB (middle panel), and with tetR and TetR-regulated sspB (right panel) were inoculated to an optical density of 0.02 without (closed figures) or with 100 ng/ml atc (open figures). Optical density was measured at the indicated time points. Data in (A), (C) and (E) are means ± SD of four replicate cultures. The four replicate cultures in (A) and (C) were pooled to prepare lysates for immunoblotting.
Figure 4.
Figure 4.
Involvement of ClpX in SspB-mediated degradation of LuxAB-DAS+4. (A) M. smegmatis luxAB-DAS+4 was transformed with an episomally replicating plasmid that expresses ClpX-K127R under the control of a TetR-regulated promoter. Bacteria in logarithmic growth were incubated at 37°C for 3 days on 7H11 agar with an atc-free (left) or an atc-containing paper disc (right). (B) Relative luminescence units of M. smegmatis luxAB-DAS+4 with constitutively expressed sspB and either a control plasmid (closed circles), TetR-regulated WT clpX (crossed circles) or TetR-regulated dominant-negative clpX (open circles) were cultured with different concentrations of atc. M. smegmatis luxAB-DAS+4 without sspB (squares) served as control for constitutive luciferase activity. Data are means ± SD of four or eight replicate cultures.
Figure 5.
Figure 5.
Effect of constitutive and TetR-regulated SspB on GFP-DAS+4 in M. tuberculosis. (A) Relative fluorescence units of M. tuberculosis gfp, M. tuberculosis gfp-ssrAec and M. tuberculosis gfp-DAS+4. White bars indicate strains without SspB; hatched bars indicate strains with a constitutively expressed SspB. n/d: not determined. (B) GFP and SspB levels detected by immunoblotting in the same strains as (A). DlaT is used as a loading control for each immunoblot. (C) Relative fluorescence units of M. smegmatis gfp-DAS+4 without sspB, with constitutively expressed sspB, or with tetR and TetR-regulated sspB. White bars indicate strains cultured in the absence of atc; hatched bars indicate those cultured in the presence of atc. (D) Relative fluorescence units of M. tuberculosis expressing untagged or variously tagged RFP, as in Figure 1C. (E) Comparison of relative fluorescence units of M. smegmatis and M. tuberculosis expressing RFP with different tags as in Figures 1C and 4D. Closed black circles indicate strains without SspB; open red circles indicate those with constitutively expressed SspB. A linear relationship is observed between RFU of M. smegmatis expressing a tagged RFP (x-axis) and RFU of M. tuberculosis expressing the same tagged RFP (y-axis). Data in (A), (C) and (D) are means ± SD of 12–20 replicates from two or three independent experiments.
Figure 6.
Figure 6.
Controlled inactivation of RpoB in M. smegmatis. (A) M. smegmatis in which the 3′-end of the chromosomal rpoB gene encodes a FLAG epitope and the DAS+4 tag was transformed with a plasmid that integrates tetR and TetR-regulated sspB into the chromosome (MR-sspB) or a control plasmid that does not contain TetR or SspB (MR-control). MR-control (left half) and MR-sspB (right half) in logarithmic growth were plated on 7H11 agar with antibiotics. Atc (0.5, 5 or 50 ng) was added to a sterile disc in the center of the plate. Plates were incubated at 37°C for 2 days. (B) Top: MR-control (triangles) and MR-sspB (circles) were inoculated to an optical density of 0.02 in media without (closed symbols) or with (open symbols) 100 ng/ml atc. Bottom: MR-control complemented with untagged rpoB (diamonds) and MR-sspB complemented with untagged rpoB (squares) were inoculated to an optical density of 0.02 in media without (closed symbols) or with (open symbols) 100 ng/ml atc. Optical density was measured at the indicated time points. (C) Left: RpoB levels detected by immunoblotting with anti-FLAG antibody from MR-sspB cultured with or without 100 ng/ml atc for 24 h. Proteasome β subunit (PrcB) serves as a loading control. Right: two-fold dilutions of the protein lysate from MR-sspB − atc cultures were loaded in comparison to that from MR-sspB + atc for semi-quantification. Data in (B) are means ± SD of four replicate cultures.
Figure 7.
Figure 7.
Susceptibility of M. smegmatis MR-sspB to rifampin and atc. (A) MR-sspB in logarithmic growth was plated on 7H11 agar containing 0, 0.05 and 0.1 ng/ml atc (second to fourth plates). Clockwise from the top, 125, 62.5 and 31.25 µg of rifampin were placed on three sterile discs on the plate. Plates were incubated at 37°C for 2–3 days. MR-control on 7H11 agar containing no atc (first plate) was also incubated with rifampin as a control. (B) MR-sspB in logarithmic growth was diluted to an optical density of 0.05 in 7H9 containing 0–0.19 ng/ml atc and incubated at 37°C with shaking for 4 days. After the pre-incubation with atc only, cultures were diluted to an optical density of 0.05 in 7H9 with the same atc concentrations but also containing 0–100 µg/ml rifampin. Optical density was measured after 4 days of incubation. Data are means ± SD of four replicates. (C) CFU recovered from MR-sspB after 0, 1 and 3 days in 0–100 ng/ml atc. Data are means ± SD of four replicate cultures.
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