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. 2008 Nov;70(4):1012-25.
doi: 10.1111/j.1365-2958.2008.06467.x. Epub 2008 Sep 22.

Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP

Kevin L Griffith  1 , Alan D Grossman
Affiliations

Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP

Kevin L Griffith et al. Mol Microbiol. 2008 Nov.

Abstract

The ability to manipulate protein levels is useful for dissecting regulatory pathways, elucidating gene function and constructing synthetic biological circuits. We engineered an inducible protein degradation system for use in Bacillus subtilis based on Escherichia coli and Caulobacter crescentusssrA tags and SspB adaptors that deliver proteins to ClpXP for proteolysis. In this system, modified ssrA degradation tags are fused onto the 3' end of the genes of interest. Unlike wild-type ssrA, these modified tags require the adaptor protein SspB to target tagged proteins for proteolysis. In the absence of SspB, the tagged proteins accumulate to near physiological levels. By inducing SspB expression from a regulated promoter, the tagged substrates are rapidly delivered to the B. subtilis ClpXP protease for degradation. We used this system to degrade the reporter GFP and several native B. subtilis proteins, including, the transcription factor ComA, two sporulation kinases (KinA, KinB) and the sporulation and chromosome partitioning protein Spo0J. We also used modified E. coli and C. crescentus ssrA tags to independently control the degradation of two different proteins in the same cell. These tools will be useful for studying biological processes in B. subtilis and can potentially be modified for use in other bacteria.

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Figures

Fig. 1
Fig. 1. Function of SspB and altered ssrA tags from E. coli in B. subtilis
A. Sequence of ssrA tags from E. coli, C. crescentus, and B. subtilis. The ClpX recognition sequence is in a box with dashed lines. The known (E. coli, C. crescentus) and putative (B. subtilis) sequence recognized by SspB is in a box with solid lines. The “A” in parenthesis is the first residue of the tag (A1) and is added onto the nascent polypeptide by the tRNA function. The other residues are encoded by the mRNA function of ssrA (tmRNA). B. Accumulation of various GFP-(Ec)ssrA mutants with and without expression of (Ec)SspB. The (Ec)ssrA tags had mutations in the C-terminal four residues (the ClpX recognition region) and the sequence is indicated above each pair of lanes. Each tag also had the +4 linker (SENY) separating the SspB-recognition sequence (AANDENY) and the four 4 C-terminal residues. Cells were grown to mid-exponential phase (OD600 ~0.2) in LB medium, at which time the culture was divided into two: one portion was left untreated (-) and the other was treated with 1 mM IPTG (+) to induce expression of Pspank-(Ec)sspB. After incubation for 1 hr, cells were harvested and the accumulation of GFP determined by Western blot with anti-GFP antibodies. The tag ending in -ALGG+4 (lanes 51-52) was used for further characterization and is designated as (Ec)ssrA*. The entire sequence of the ssrA* tag is: -AANDENYSENYALGG. Strains with each tag are listed in Table 1. C. Kinetics of degradation of GFP-(Ec)ssrA* and ComA-(Ec)ssrA* after induction of Pspank-(Ec)sspB. Strains KG951 (GFP-(Ec)ssrA*) and KG1060 (ComA-(Ec)ssrA*) were grown in LB medium to mid-exponential phase (OD600 of ~0.5) and IPTG was added to induce expression of Pspank-(Ec)sspB. The amounts of GFP-(Ec)ssrA* (top panel) and ComA-(Ec)ssrA* (bottom panel) were determined by Western blot with anti-GFP or anti-ComA antibodies before (time 0) and at the indicated times (5, 10, 15, 20, 30 min for GFP; and 2, 4, 6, 8, 10 min for ComA) after addition of IPTG. D. Degradation of GFP-(Ec)ssrA* depends on clpX but not clpC. Strains containing GFP-(Ec)ssrA* and Pspank-(Ec)sspB and either a null mutation in clpX (KG1185) or a null mutation in clpC (KG946) or wild type (KG951) were grown in LB medium. The amount of GFP was determined by Western blot in untreated cells or 60 min after treatment with IPTG to induce production of (Ec)SspB.
Fig. 2
Fig. 2. Effects of ssrA degradation tags and basal expression of SspB on ComA activity
Cells containing PsrfA-lacZ were grown in defined minimal medium and transcription of srfA-lacZ was monitored by measuring β-galactosidase specific activity at times throughout growth. Where indicated, 1 mM IPTG was added to cultures at the time of inoculation to induce expression of Pspank-(Ec)-sspB. β-galactosidase specific activity is plotted as a function of cell density (OD600). The effects of ssrA degradation tags on ComA activity in the absence of any adaptor protein; KG1343 (wild type, no ssrA tag; open diamonds); KG1205 (ComA-(Ec)ssrA-ALGG+4 (ssrA*); “X”); and KG1235 (ComA-(Ec)ssrA-ADAN+4; dashed line). The effects of basal (uninduced) expression from Pspank-(Ec)sspB on ComA-(Ec)ssrA* activity: KG1178 (wild type Pspank-(Ec)sspB) without IPTG, filled squares; with IPTG, open squares. KG1180 (Pspank(T-35C)-(Ec)sspB) without IPTG, black triangles; with IPTG, open triangles. KG1177 (Pspank(T-7C)-(Ec)sspB) without IPTG, black circles; with IPTG open circles.
Fig. 3
Fig. 3. Specificity of SspB and ssrA from E. coli and C. crescentus
Cells containing the indicated GFP-ssrA (A, B) or ComA-ssrA (B) fusions and either (Ec)sspB or (Cc)sspB fused to an inducible promoter were grown in LB medium to mid-exponential phase and left untreated or treated with inducer (IPTG or xylose) for one hour. The amount of GFP or ComA was then measured by Western blot. A. Cells expressed GFP fused to the indicated variant of E. coli (Ec) ssrA or C. crescentus (Cc) ssrA. Cells also contained Pspank fused to either (Cc)sspB (top bands) or (Ec)sspB (bottom bands). The pairs of strains ((Cc)sspB strains indicated first and (Ec)sspB second) with each ssrA variant are: KG1023 / KG951, (Ec)ssrA* (lanes 1-2); KG1024 / KG845, (Ec)ssrA-ALDD+4 (lanes 3-4); KG1025 / KG1018, (Cc)ssrA-ALGG (lanes 5-6); KG1026 / KG1019, (Cc)ssrA-ALDD (lanes 7-8); KG1027 / KG1020, (Cc)ssrA(A1W)-ALGG (lanes 9-10); and KG1028 / KG1021, (Cc)ssrA(A2D)-ALGG (lanes 11-12). B. Dual tagging and degradation system. Strain KG1201, containing Pspank-(Cc)sspB and Pxyl-(Ec)sspB, and ComA-(Cc)ssrA(A2D)-ALGG and GFP-(Ec)ssrA*, was left untreated or treated with IPTG, or xylose, or both for 1 hr. Cultures were harvested and the accumulation of GFP and ComA determined by Western blot. ComA-(Cc)ssrA(A2D)-ALGG is degraded only when (Cc)sspB is expressed and GFP-(Ec)ssrA* is degraded only when (Ec)sspB is expressed.
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