title: Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase.
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.
authors.affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.
authors.forename: Jee-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Jun-Rong
authors.initials: JR
authors.lastname: Wei
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Wallach
authors.forename: Rebekkah S
authors.initials: RS
authors.lastname: Robbins
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
authors.forename: Dirk
authors.initials: D
authors.lastname: Schnappinger
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000235:genetics; D055550:Protein Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D012329:RNA, Bacterial; C477700:sspB protein, E coli; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D010447:Peptide Hydrolases