Functional genomic characterization of metallothioneins in brown trout (Salmo trutta L.). using synthetic genetic analysis

Abstract

Metal pollution has made a significant impact on the earth's ecosystems and tolerance to metals in a wide variety of species has evolved. Metallothioneins, a group of cysteine-rich metal-ion binding proteins, are known to be a key physiological mechanism in regulating protection against metal toxicity. Many rivers across the southwest of England are detrimentally affected by metal pollution, but brown trout (Salmo trutta L.) populations are known to reside within them. In this body of work, two isoforms of metallothionein (MetA and MetB) isolated from trout occupying a polluted and a control river are examined. Using synthetic genetic array (SGA) analyses in the model yeast, Saccharomyces cerevisiae, functional genomics is used to explore the role of metallothionein isoforms in driving metal tolerance. By harnessing this experimental system, S. cerevisiae is used to (i) determine the genetic interaction maps of MetA and MetB isoforms; (ii) identify differences between the genetic interactions in both isoforms and (iii) demonstrate that pre-exposure to metals in metal-tolerant trout influences these interactions. By using a functional genomics approach leveraged from the model yeast Saccharomyces cerevisiae, we demonstrate how such approaches could be used in understanding the ecology and evolution of a non-model species.

Figure 1

Map of River locations in Britain. Map of Britain, highlighting the rivers Hayle and Camel in southwest England. The River Hayle was used in this study as a region that has suffered from historical metal pollution. The River Camel was used as a relatively clean source in comparison. DNA from trout from both rivers was used to amplify the MetA and MetB genes.

Figure 2

(A) Metal tolerant phenotypic screen. S. cerevisiae expressing one of the four genes MetA_Cam, MetB_ Cam, MetA_Hay and MetB_Hay) or an empty plasmid were tenfold serially diluted onto agar plates containing sub-lethal and lethal levels of metals (Table 1). The plates were incubated for 2 days at 30 °C. All of the MT genes amplified from the fish resulted in some level of metal tolerance; however, the most dramatic effects can be observed through the expression of MetB isolated from trout in the River Hayle. 1: S. cerevisiae transformed with empty plasmid p426_ccBd; 2: S. cerevisiae transformed with a plasmid containing MetA isolated from River Camel trout; 3: S. cerevisiae transformed with a plasmid containing the MetA gene from River Hayle trout; 4: S. cerevisiae transformed with a plasmid containing the MetB gene from River Camel trout; 5: S. cerevisiae transformed with a plasmid containing the MetB gene from River Hayle trout. (B) Levels of expression of MetA and MetB from the rivers Camel and Hayle when expressed in S. cerevisiae.

Figure 3

Genetic interaction network of MetA and MetB from Camel and Hayle trout. Genome-wide synthetic interaction SGA screens were performed using query strains that were transformed with (A) MetA_Cam, MetA_Hay and (B) MetB_Cam, MetB_Hay. Genes are represented by nodes that are colour-coded according to their Saccharomyces Genome Database (SGD) cellular roles (www.yeastgenome.org). Interactions are represented by edges. Deletion mutants that display a synthetic lethal (SL) interaction are detailed in Supplemental Table 2.