Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP.
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.
Griffith
Kevin L
KL
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Grossman
Alan D
AD
D000483:Alleles; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D016935:Caulobacter crescentus / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; C061262:ComA protein, Bacteria; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C477700:sspB protein, E coli; C096222:tmRNA; D049452:Green Fluorescent Proteins; D049071:Endopeptidase Clp