Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression.
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.
Triccas
James A
JA
Centenary Institute of Cancer Medicine and Cell Biology, Newtown NSW, Australia. J.Triccas@cententary.usyd.edu.au
Pinto
Rachel
R
Britton
Warwick J
WJ
D003001:Cloning, Molecular; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009161:Mycobacterium / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014169:Transformation, Bacterial
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins