Green fluorescent protein as a new expression marker in mycobacteria

Mol Microbiol. 1995 Sep;17(5):913-22. doi: 10.1111/j.1365-2958.1995.mmi_17050913.x.


This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antitubercular Agents / pharmacology
  • Base Sequence
  • Cell Line
  • Chaperonin 60 / biosynthesis
  • Chaperonin 60 / genetics
  • DNA Primers
  • Genetic Vectors
  • Green Fluorescent Proteins
  • Luminescent Proteins / analysis
  • Luminescent Proteins / biosynthesis*
  • Luminescent Proteins / genetics
  • Macrophages / microbiology*
  • Mice
  • Microbial Sensitivity Tests
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mycobacterium / drug effects
  • Mycobacterium / isolation & purification
  • Mycobacterium / physiology*
  • Mycobacterium bovis / drug effects
  • Mycobacterium bovis / isolation & purification
  • Mycobacterium bovis / physiology*
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Recombinant Proteins / analysis
  • Recombinant Proteins / biosynthesis


  • Antitubercular Agents
  • Chaperonin 60
  • DNA Primers
  • Luminescent Proteins
  • Recombinant Proteins
  • Green Fluorescent Proteins