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Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression


Blokpoel, MCJ and O'Toole, R and Smeulders, MJ and Williams, HD, Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression, Journal of Microbiological Methods, 54, (2) pp. 203-211. ISSN 0167-7012 (2003) [Refereed Article]

DOI: doi:10.1016/S0167-7012(03)00044-7


Unstable variants of green fluorescent protein (GFP) tagged with C-terminal extensions, which are targets for a tail specific protease, have been described in Escherichia coli and Pseudomonas putida [Appl. Envir. Microbiol. 64 (1998) 2240]. We investigated whether similar modifications to flow cytometer optimised GFP (GFPmut2) could be used to generate unstable variants of GFP for gene expression studies in mycobacteria. We constructed GFP variants in a mycobacterial shuttle vector under the control of the regulatory region of the inducible Mycobacterium smegmatis acetamidase gene. GFP expression was induced by the addition of acetamide and the stability of the GFP variants in M. smegmatis, following the removal of the inducer to switch off their expression, was determined using spectrofluorometry and flow cytometry. We demonstrate that, compared to the GFPmut2 (half-lives>7 days), the modified GFP variants exhibit much lower half-lives (between 70 and 165 min) in M. smegmatis. To investigate their utility in the measurement of mycobacterial gene expression, we cloned the promoter region of a putative amino acid efflux pump gene, lysE (Rv1986), from Mycobacterium tuberculosis together with the divergently transcribed, putative lysR-type regulator gene (Rv1985c) upstream of one of the unstable GFP variants. We found that the expression kinetics of the lysRE-gfp fusion were identical throughout the M. smegmatis growth curve to those measured using a conventional lysRE-xylE reporter fusion, peaking upon entry into stationary phase. In addition, it was established that the tagged GFP variants were also unstable in Mycobacterium bovis BCG. Thus, we have demonstrated that unstable GFP variants are suitable reporter genes for monitoring transient gene expression in fast- and slow-growing mycobacteria.

Item Details

Item Type:Refereed Article
Keywords:Acetamidase; Flow cytometry; LysE; LysR; Mycobacterium tuberculosis; Stationary phase
Research Division:Biomedical and Clinical Sciences
Research Group:Medical microbiology
Research Field:Medical bacteriology
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:O'Toole, R (Dr Ronan O'Toole)
ID Code:95794
Year Published:2003
Web of Science® Times Cited:29
Deposited By:Medicine
Deposited On:2014-10-08
Last Modified:2014-10-08

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