Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulato
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O RI G I NAL PAPE R
Tetracycline-inducible gene expression in mycobacteria within an animal host using modiWed Streptomyces tcp830 regulatory elements S. Moises Hernandez-Abanto · Samuel C. Woolwine · Sanjay K. Jain · W. R. Bishai
Received: 6 June 2006 / Revised: 21 July 2006 / Accepted: 25 July 2006 / Published online: 30 August 2006 © Springer-Verlag 2006
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 tetracyclineinducible 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 modiWed to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green Xuorescent 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. Keywords Anhydrotetracycline · Acetamide · Mycobacteria
S. M. Hernandez-Abanto · S. C. Woolwine · S. K. Jain · W. R. Bishai (&) 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 e-mail: [email protected]
Introduction Tuberculosis is currently the seventh leading cause of disability and death globally and is expected to remain so if current tools and patterns of control prevail (Murray and Salomon 1998). The 2004 World Health Organization (WHO) report on global TB control estimates that 8.8 million people acquire new tuberculosis disease each year (WHO 2004). One of the major reasons for the success of Mycobacterium tuberculosis as a pathogen is its ability to adapt to a wide range of conditions inside the human host. This includes its ability to stay dormant in a latent phase in the host for years or even decades. WHO estimates that one-third of the world’s population is latently infected with M. tuberculosis, and many develop reactivation disease, years after the initial exposure (Lurie 1942; Stead 1967). The mechanisms that allow M. tuberculosis to adapt to these wide ranges of conditions and/or reactivate are not clearly understood. Studying mycobacterial genes involved in this adaptive response is essential to understand the mechanisms involved in latency and other mycobacterial adaptive responses. Inducible expression systems are powerful tools f
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