Green fluorescent protein as a new expression marker in mycobacteria.

Abstract:

: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.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Kremer L,Baulard A,Estaquier J,Poulain-Godefroy O,Locht C

doi

10.1111/j.1365-2958.1995.mmi_17050913.x

subject

Has Abstract

pub_date

1995-09-01 00:00:00

pages

913-22

issue

5

eissn

0950-382X

issn

1365-2958

journal_volume

17

pub_type

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