Abstract
Gas vesicle formation of Halobacterium salinarum is regulated by the transcriptional activator GvpE, and in the presence of the repressing protein GvpD, the amount of GvpE is strongly reduced. The green fluorescence protein was used to report this GvpD-mediated reduction of GvpE in vivo in Haloferax volcanii transformants. Both N- or C-terminal fusions of GFP to GvpE were tested, but only the N-terminal fusion reported the reduction. The fluorescence of GFP-GvpE was 62 % reduced with GvpD wild type (DWT), 78 % with the super-repressor D3-AAA, and only 10 % with the repression defect DMut6. Further analysis of D3-AAA indicated that the super-repression was due to the alteration R496A. GFP-GvpE variants defect in promoter activation was tested in the presence of DWT, D3-AAA and DMut6, and two of them were more stable. Overall, the GFP-GvpE fusion was suitable to study and quantify the amount of GvpE in vivo.
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Acknowledgments
We wish to thank Julie Maupin-Furlow (Gainesville, Florida, USA) for plasmid pJAM1020 expressing smRS-GFP, and Sabrina Fröls (TU Darmstadt, Germany) for help in fluorescence microscopy. This work received financial support by the German Research Council, DFG (Pf165/10-1).
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Schmidt, I., Pfeifer, F. Use of GFP-GvpE fusions to quantify the GvpD-mediated reduction of the transcriptional activator GvpE in haloarchaea. Arch Microbiol 195, 403–412 (2013). https://doi.org/10.1007/s00203-013-0885-7
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DOI: https://doi.org/10.1007/s00203-013-0885-7