title: Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects.
abstract: The significance of metal(oid)s as environmental pollutants has made them a priority in ecotoxicology, with the aim of minimizing exposure to animals or humans. Therefore, it is necessary to develop sensitive and inexpensive methods that can efficiently detect and monitor these pollutants in the environment. Conventional analytical techniques suffer from the disadvantages of high cost and complexity. Alternatively, prokaryotic or eukaryotic whole-cell biosensors (WCB) are one of the newest molecular tools employed in environmental monitoring that use the cell as an integrated reporter incorporating a reporter gene fused to a heavy metal responsive promoter. In the present paper, we report results from expressing, in the ciliate Tetrahymena thermophila, constructs consisting of the reporter gfp gene fused to the complete MTT1 or MTT5 protein coding regions under the transcriptional control of the MTT1 metallothionein promoter, which plays a critical role in heavy metal stress in this ciliate. When exposed to Cd(2+), such cells overexpress both the GFP reporter transgene and the linked metallothionein gene. We report that, for the GFPMTT5 strain, this metallothionein overexpression results in marked resistance to cadmium toxicity (24 h LC50 ~15 μM of Cd(2+)), compared to wild type cells (24 h LC50 ~1.73 μM of Cd(2+)). These results provide the first experimental evidence that ciliate metallothioneins, like in other organisms, function to protect the cell against toxic metal ions. Because these strains may have novel advantages as WCBs, we have compared their properties to those of other previously reported Tetrahymena WCBs.
authors.affiliation: Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense (UCM), C/. José Antonio Novais 12, 28040, Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Amaro
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Turkewitz
authors.forename: Ana
authors.initials: A
authors.lastname: Martín-González
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Gutiérrez
mesh_terms: D015374:Biosensing Techniques; D002104:Cadmium / Q000032:analysis*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012989:Soil Pollutants / Q000032:analysis; D016808:Tetrahymena thermophila / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D012989:Soil Pollutants; D002104:Cadmium; D049452:Green Fluorescent Proteins; D008668:Metallothionein