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Infection and Immunity, March 2003, p. 1379-1388, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1379-1388.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology

Beatrice Saviola,1,{dagger} Samuel C. Woolwine,2 and William R. Bishai2,3*

Department of Molecular Microbiology and Immunology,1 Department of International Health, Johns Hopkins Bloomberg School of Public Health,3 Division of Infectious Diseases, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland2

Received 23 September 2002/ Returned for modification 23 October 2002/ Accepted 21 November 2002

A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon {gamma}{delta} is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.

* Corresponding author. Mailing address: Division of Infectious Diseases, Dept. of Medicine, Center for Tuberculosis Research, Johns Hopkins School of Medicine, 424 N. Bond St., Baltimore, MD 21231-1001. Phone: (410) 955-3507. Fax: (410) 614-8173. E-mail: wbishai{at}jhsph.edu.

Editor: J. N. Weiser

{dagger} Present address: Department of Microbiology, School of Osteopathic Medicine, Western University, Pomona, CA 91766-1854.

Infection and Immunity, March 2003, p. 1379-1388, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1379-1388.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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