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 Cd2+, 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 (24h LC50 ~ 15 µM of Cd2+), compared to wild type cells (24h LC50 ~ 1.73 µM of Cd2+). 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.

Genome plasticity in response to stress inTetrahymena thermophila: selective and reversible chromosome amplification and paralogous expansion of metallothionein genes

“…CU428 was used to obtain KO and KD strains (MTT1KO, MTT5KD and MTT1KO 1 MTT5KD), as has been described in de Francisco et al,(2017). GFPMTT1 bears a multicopy plasmid encoding the MTT1 gene (Amaro et al, 2014). The three metal-adapted strains (Cd-adap, Cu-adap and Pb-adap) were obtained after extended culture in metal-containing media (De Francisco et al, 2017).…”

Biosensors and their Applications in Food Safety: A Review

“…For example, microbial fluorescence-based biosensor devices (Tao et al, 2013;Amaro et al, 2014) use reporter genes that react only when biochemical interactions occur between cellular reporters and inducer molecules. A combination of a chemostat-like microfluidic platform and microbial biosensors facilitates molecular analyte detection on a chip (Kim et al, 2015).…”

Light dazzles from the black box: whole-cell biosensors are ready to inform on fundamental soil biological processes

“…However, while microalgae and yeast-based biosensors have been mainly used for studying metal availability in water bodies and other environmental matrices, protozoa biosensors such as Tetrahymena thermophila inserted with the luc or gfp genes in transcriptional fusion with metallothionein genes inducible upon exposure to Cd and Pb showed promising applications [3,4].…”