Genomic, transcriptomic and physiological analyses of silver‐resistant Saccharomyces cerevisiae obtained by evolutionary engineering

Abstract

Silver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO 3 in 29 populations and obtained highly silver-resistant and genetically stable Saccharomyces cerevisiae strains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in the RLM1 gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistant S. cerevisiae strain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast.

Fungal–Metal Interactions: A Review of Toxicity and Homeostasis

“…It was previously described that exposure to AgNO 3 and Ag NPs resulted in the increased expression of CUP1-1 and CUP1-2 , proposing that the encoded MTs may also bind Ag + and decrease sensitivity [ 81 , 114 , 115 ]. Similar results were observed in AgNO 3 exposure, where yeast had increased expression of CUP1-1 and CUP1-2 (4.79-fold and 4.71-fold, respectively) in an extended study that resulted in an evolved yeast strain, confirming the potential role of copper MTs in silver resistance [ 116 ]. Other Ag + transporters, Pho84, Fet3, and Smf1, were not implicated in Ag + uptake; however, significant down regulation (68.56-fold) of PHO84 in silver evolved yeast has been observed, which may indicate that Pho84 plays a role in Ag + uptake, and may serve as a mechanism of Ag + resistance [ 81 , 116 ].…”

“…Silver is a non-essential metal that has no designated cellular receptors or membrane channels for ion uptake. Much of the literature has focused on silver as an antimicrobial agent, but some studies have begun to clarify homeostatic mechanisms [ 81 , 114 , 116 , 298 ]. Silver has properties similar to copper, which has initiated the evaluation of copper homeostatic systems to investigate how they may contribute to silver uptake and transport [ 21 , 81 , 114 , 116 , 298 ].…”

“…The worldwide increase of silver usage makes studies on mechanisms of silver resistance important; presently, few studies have reported on this. CTR3 is implicated in Ag + resistance after an observed fold increase in its expression in a silver evolved strain of S. cerevisiae [ 116 ]. Insight into the expression of the Ctr3 transcription factor MAC1 in the presence of Ag + may clarify its role in resistance.…”

“…Other Ag + transporters, Pho84, Fet3, and Smf1, were not implicated in Ag + uptake; however, significant down regulation (68.56-fold) of PHO84 in silver evolved yeast has been observed, which may indicate that Pho84 plays a role in Ag + uptake, and may serve as a mechanism of Ag + resistance [ 81 , 116 ]. The effect of Ag + on genes involved in ergosterol biosynthesis was also investigated in a silver evolved yeast [ 116 ]. Results indicated down-regulation of those genes, suggesting that one mechanism of action of resistance against Ag + toxicity could be the ability to inhibit their down regulation [ 116 ].…”

“…The effect of Ag + on genes involved in ergosterol biosynthesis was also investigated in a silver evolved yeast [ 116 ]. Results indicated down-regulation of those genes, suggesting that one mechanism of action of resistance against Ag + toxicity could be the ability to inhibit their down regulation [ 116 ]. In the filamentous fungus A. nidulans , silver induced expression of copper exporter crpA, indicating that it may play a role in silver export and resistance [ 90 ].…”