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Journal of Bacteriology, May 2000, p. 2732-2740, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552'phoA In Vitro Transposition System

Miriam Braunstein,1 Thomas J. Griffin IV,2 Jordan I. Kriakov,1 Sarah T. Friedman,1 Nigel D. F. Grindley,2 and William R. Jacobs Jr.1,*

Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461,1 and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-81142

Received 9 December 1999/Accepted 22 February 2000

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

* Corresponding author. Mailing address: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-2888. Fax: (718) 518-0366. E-mail: jacobs{at}aecom.yu.edu.

Journal of Bacteriology, May 2000, p. 2732-2740, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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