Global transcriptome and deletome profiles for yeast exposed to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium) and VB (arsenic) elements were generated and compared. Genomic data sets were generated using an experimental design to minimize variations associated with differences in yeast strain backgrounds, microarray platforms, and data extraction and analysis techniques. In addition, equi-toxic concentrations of metals were used to minimize differences due to variations in the level of toxicity. Several studies have shown that minimizing experimental variation is essential for decreasing statistical variability in microarray data 
. Thus, by minimizing experimental variations, transcriptional responses associated primarily with the different metals were identified.
Several studies have compared the effects of exposure to multiple metals on the transcriptome in yeast and mammalian cells. There was, however, little overlap between the responsive genes reported in these studies and those presented in the current report. When enriched GO categories were compared however, genes involved in detoxification of oxidative stress commonly respond to stress conditions 
Transcriptome analysis revealed that each of the seven metals affected the level of expression of 175–760 genes (). PCA and hierarchical clustering by experimental conditions grouped the expression patterns by physiological/chemical characteristics of the metals. PCA and clustering placed cadmium and mercury, two non-essential metals, close together and separate from the essential metals; zinc, chromium, and copper. Silver closely grouped with zinc. This may be because silver can substitute for other metals in many biological activities in yeast and may be sensed as an essential metal 
. Although chromium is essential, it is a Group VIB transition metal with different chemical properties compared to the others. These results suggest that the chemical properties of the metal/metalloid contribute to the pattern of the genomic response.
K-means clustering illustrated that the expression of ~60% of the genes was similarly affected by all seven metals. In addition, two sub-sets of metals: (A) zinc, chromium, mercury, silver and cadmium; and (B) arsenic and copper, affected the expression of similar genes. This suggests that cells may have developed common or convergent transcription regulatory mechanisms to accommodate metal exposure. Copper and arsenic, which undergo redox reactions in vivo, affected the expression of similar genes. This suggests that the ability to participate in redox reactions defines a unique set of metal-responsive genes. Although chromium is redox active in vivo, it clustered distinctly from copper and arsenic (). Several chromium responsive genes in Clusters IV and VI did, however, show expression levels similar to those of copper and arsenic. These results suggest that the ability to undergo redox reactions in vivo contributes to the global transcriptional response, but other chemical properties of the metal may dominate the response.
Generating precursor metabolites and energy, synthesizing proteins and amino acids, transporting chemicals and proteins, and responding to oxidative stress were common responses to metal exposure. Furthermore, there were increases in detoxifying processes such as metal chelation, sequestration of metals into endosomal vacuoles, and exocytosis. These responses may reduce the intracellular levels of the metal by decreasing the number of metal-importing transporters and increasing metal efflux through major facilitators. The processes identified in this study are similar to adaptations in yeast exposed to other environmental stressors, as well as in human fibroblasts exposed to arsenic 
The expression of genes associated with the production of ribosomes was also affected by metal exposure. Other environmental stresses have also been shown to repress ribosomal protein expression and the translation apparatus 
. Ribosome production may utilize more than 50% of the synthetic effort of rapidly growing eukaryotic cells 
. Therefore, by inhibiting ribosome synthesis, cells may be able to redirect these resources towards the defense against metal toxicity 
Analyses of the transcriptome and deletome identified several evolutionarily conserved signal transduction pathways that may be involved in regulating the responses to metal exposure. These include those mediated by cAMP-dependent protein kinase A (PKA), protein kinase CK2, and MAPK.
The PKA pathway coordinates post-translational regulation of a variety of proteins such as key enzymes of glycolysis and gluconeogenesis, and the transcriptional control of ribosomal protein and stress response proteins 
. Of the 1,341 metal responsive genes ~10% are stress response genes whose expression is controlled by the PKA-regulated transcription factors Msn2p/Msn4p 
. When PKA is activated, Msn2p/Msn4p activity is repressed. Non-activation of PKA due to low cAMP levels will ultimately lead to the derepression of Msn2p/Msn4p 
. The mechanism by which metals inactivate PKA has not been fully defined; however, our results suggest that metal exposure may reduce the levels of cytoplasmic glucose. In yeast, low glucose causes a decrease in the level of cAMP with concomitant low PKA activity 
. Changes in glucose levels may be the result of a combination of changes in yeast metabolism. There were decreases in the expression of high affinity hexose plasma membrane transporters (HXT2, HXT4, HXT6, HXT7
) and increases in the expression of genes associated with glycolysis and hexose metabolism (PGK1 ENO2 FBA1 TYE7/SGC1 CDC19
) (; Table S1
). Furthermore, there was a decrease in the expression of the maltase genes MAL12
, which metabolize maltose into glucose 
. The combination of decreased sugar production and transport, and increased metabolism may lead to a level of glucose that would limit PKA activity. This will ultimately lead to increased stress response gene expression via Msn2p/Msn4p.
Protein kinase CK2 functions in diverse cellular processes 
. Protein kinase CK2, along with Utp22, Rrp7 and Ifh1 comprises the CURI protein complex. It has been suggested that the CURI complex is involved in ribosome synthesis, mediating transcription and processing of pre-rRNAs, and the transcription of ribosomal protein genes 
. Metal treatment caused a decrease in CKA1
expression. This is similar to the results obtained in arsenic exposed JB6 mouse epidermal cells 
. Thus, decreased ribosome biosynthesis associated with metal exposure may be caused by the repression of protein kinase CK2, as well as decreased PKA activity 
MAPK cascades participate in various cellular processes including apoptosis, differentiation and the stress response. MAPKs have been shown to mediate inducible transcription in response to exposure of the seven metals examined in this study 
. In yeast, five MAPKs have been identified 
. Among them, HOG1
, a mammalian p38 homolog, regulates the high osmolarity glycerol response, which is activated through two independent upstream pathways that converge at MAPKK PBS2 
. The deletome analysis revealed that proteins in the high osmolarity glycerol response (Sho1, Ste20, Ssk1, Ssk2, Pbs2, Hog1) are required for arsenic, cadmium and zinc tolerance. Another MAPK cascade that regulates the cell wall construction pathway (Bck1, Mkk1) was required for cadmium tolerance. Cytoscape analysis of the transcriptome revealed that a group of proteins that interact with the MAPKKK Pkh2, a serine/threonine kinase involved in maintenance of cell wall integrity, significantly changed in their levels of expression (). These results are consistent with other studies that demonstrated a role of MAPK cascades in controlling metal-responsive transcription. Furthermore, they indicate that proper functioning of the MAPK pathways is essential for survival to environmental stresses.
The deletome was created by measuring the growth characteristics of each mutant strain in the presence of metal. Hierarchical clustering showed limited overlap in the genes comprising the different clusters of mutants exposed to the metals (). This suggests that resistance to metal toxicity may have divergent mechanisms.
Mercury and silver had the lowest number of genes in the deletome. This may be a consequence in how yeast responds to exposure of these metals. When yeast was grown in increasing concentrations of these metals, there was a concentration-dependent increase in the length of the stationary growth phases. However, exponential growth rates and maximal cell densities were similar at almost all of the mercury and silver concentrations (Figure S1
). In contrast, growth in the presence of the other metals caused concentration-dependent decreases in growth rates and maximal cell densities. Thus, yeast may better adapt to silver and mercury exposure. Exposure to metals at concentrations beyond which yeast can adapt may reveal additional genes in the deletome. Alternatively, monitoring deletion strains for metal-induced changes in the stationary phase identify other essential genes.
A comparison of genes in the transcriptome and deletome did not identify many common genes (). This lack of correlation has been observed in other studies. It may be attributed to genetic redundancy or the inability of microarrays to measure non-transcriptionally regulated changes in activity 
. The lack of overlap may be due to differences between the measured end points. For the transcriptome, RNA was purified from the cells exposed to metals for 2 hours, during lag phase of growth. Yeast begin to respond to new environments and adapt for growth during this period of time. Thus, the genes identified in the transcriptome may contribute to the early adaptation of metal-induced stress. In contrast, GIF's were calculated from yeast in stationary growth phase and may be related to cell proliferation. Many of the other yeast deletomes also examined cells that are in the stationary phase 
. In the future, a greater overlap between the transcriptome and deletome may be achieved if GIF's are calculated from metal-induced differences in lag times, initial growth rates, and maximum cell density.
By combining Gene Ontology results from the transcriptome and deletome, several common processes were identified (). These include cation and transition metal transport, and sulfur amino acid transport and biosynthesis, which were required for cell growth in the presence of arsenic, cadmium, chromium and copper. Gene Ontology results suggest that genes in the deletome may represent convergent or central upstream points in pathways, which ultimately protect cells against metal toxicity. Furthermore, the transcriptome may identify the downstream genes in the cognate pathways. For example, the deletome contained genes involved in serine and threonine metabolism; glutamate, aspartate and arginine metabolism; and shikimate metabolism. These genes are located upstream in the sulfur, methionine and homocysteine metabolic pathways. The downstream genes in these pathways were identified in the transcriptome. Changes in sulfur amino acids is consistent with results obtained in previous studies of arsenic exposure, where processes involved in glutathione synthesis overlapped in the transcriptome and deletome 
The capacity to respond to environmental stresses is critical to the survival and propagation of all organisms. Genomic studies provide important information on molecular mechanisms of environmental stress responses. In this study, we have identified genes that respond to exposure of essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanisms by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways.