title: Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology.
abstract: Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049-Madrid, Spain.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cava
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: de Pedro
authors.forename: Emilio
authors.initials: E
authors.lastname: Blas-Galindo
authors.forename: Geoffrey S
authors.initials: GS
authors.lastname: Waldo
authors.forename: Lars F
authors.initials: LF
authors.lastname: Westblade
authors.forename: José
authors.initials: J
authors.lastname: Berenguer
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006358:Hot Temperature; D008164:Luminescent Proteins / Q000378:metabolism; D008828:Microbiological Techniques / Q000379:methods*; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins