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

TitleProtein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase.
Publication TypeJournal Article
Year of Publication2011
AuthorsKim J-H, Wei J-R, Wallach JB, Robbins RS, Rubin EJ, Schnappinger D
JournalNucleic Acids Res
Date Published2011 Mar
KeywordsBacterial Proteins, Carrier Proteins, DNA-Directed RNA Polymerases, Enzyme Stability, Escherichia coli Proteins, Kinetics, Luminescent Proteins, Mutation, Mycobacterium smegmatis, Mycobacterium tuberculosis, Peptide Hydrolases, Protein Stability, RNA, Bacterial

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.

Alternate JournalNucleic Acids Res
PubMed ID21075796
PubMed Central IDPMC3064785
Grant ListP01 AI68135 / AI / NIAID NIH HHS / United States

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