• Open Access

Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Authors

  • Sougata Roy,

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India.
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    • Present addresses: Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94158, USA;

  • Yeddula Narayana,

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India.
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    • Laboratory of Genetics, Salk Institute for Biological Studies, San Diego, California, USA.

  • Kithiganahalli Narayanaswamy Balaji,

    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India.
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  • Parthasarathi Ajitkumar

    Corresponding author
    1. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore – 560012, Karnataka, India.
      E-mail ajit@mcbl.iisc.ernet.in; Tel. (+91) 80 2293 2344; Fax (+91) 80 2360 2697.
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E-mail ajit@mcbl.iisc.ernet.in; Tel. (+91) 80 2293 2344; Fax (+91) 80 2360 2697.

Summary

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfpm2+ gene, coding for GFPm2+ of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

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