title: Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements.
abstract: Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The -10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.
authors.affiliation: Center for Tuberculosis Research, Department of Medicine, Division of Infectious Diseases, Johns Hopkins School of Medicine, 1550 Orleans Street, Rm.1.08, Baltimore, MD 21231-1044, USA.
authors.forename: S Moises
authors.initials: SM
authors.lastname: Hernandez-Abanto
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Woolwine
authors.forename: Sanjay K
authors.initials: SK
authors.lastname: Jain
authors.forename: W R
authors.initials: WR
authors.lastname: Bishai
mesh_terms: D000081:Acetamides / Q000494:pharmacology; D000818:Animals; D051379:Mice; D009165:Mycobacterium Infections, Nontuberculous / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional / Q000502:physiology; D012097:Repressor Proteins / Q000502:physiology*; D048372:Streptomyces coelicolor / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D013754:Tetracyclines / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D000081:Acetamides; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C016229:4-epianhydrotetracycline; C030686:acetamide; D013752:Tetracycline