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Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression
James A Triccas1, Rachel Pinto, Warwick J Britton
1Centenary Institute of Cancer Medicine and Cell Biology, Newtown NSW, Australia. J.Triccas@cententary.usyd.edu.au
Abstract:
The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells. The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression. We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria. By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E. coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependent degradation within the fast-growing species Mycobacterium smegmatis. This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress. Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria. This system will facilitate studies exploring dynamic changes in mycobacterial gene expression.
Insights
Researchers created a destabilized green fluorescent protein (GFP) to track rapid gene expression changes in mycobacteria. This new tool, unlike standard GFP, effectively monitors transient gene activity, advancing mycobacterial research.
Area of Science:
- Molecular Biology
- Microbiology
- Biochemistry
Background:
- Green fluorescent protein (GFP) is vital for studying gene expression and protein localization in living cells.
- The inherent stability of GFP limits its utility for detecting transient gene expression changes.
- Monitoring dynamic gene expression in mycobacteria presents unique challenges due to existing reporter limitations.
Purpose of the Study:
- To develop a destabilized variant of GFP (dGFP) for monitoring dynamic gene expression in mycobacteria.
- To assess the utility of dGFP in detecting transient changes in gene activity, particularly in response to stress.
- To investigate the differential stability of dGFP in fast-growing versus slow-growing mycobacterial species.
Main Methods:
- A destabilized form of GFP was engineered by fusing an 11-amino acid peptide from the E. coli ssrA gene to the C-terminus of GFP.
- The engineered dGFP was expressed in Mycobacterium smegmatis (fast-growing) and Mycobacterium bovis bacille Calmette-Guerin (BCG, slow-growing).
- The ability of dGFP and standard GFP to report on the activity of the stress-induced Mycobacterium tuberculosis sigE promoter under acid stress conditions was compared.
Main Results:
- The destabilized GFP (dGFP) demonstrated gradual, time-dependent degradation in Mycobacterium smegmatis, enabling detection of transient gene expression changes.
- Unmodified GFP showed a delayed response to acid stress, failing to capture early, transient promoter activity.
- Both GFP and dGFP exhibited similar stability in the slow-growing BCG strain, indicating species-specific recognition of the ssrA tag.
Conclusions:
- The developed destabilized GFP provides a valuable tool for monitoring dynamic and transient gene expression in fast-growing mycobacteria.
- This system overcomes the limitations of stable GFP, facilitating more nuanced studies of gene regulation in response to environmental cues.
- Differential stability of the ssrA tag in slow- versus fast-growing mycobacteria highlights species-specific regulatory mechanisms and offers new avenues for research.