2.1 Media and growth condition
Escherichia coli strain XL1-Blue was routinely grown at 370C in YT medium (0.5% yeast extract, 1% tryptone, 0.5% sodium chloride). 100µg Ampicillin ml-1 was added for isolation of the plasmid. YPD (1% yeast extract, 2% peptone, 2% dextrose) and SD (synthetic dextrose) media were prepared as described. 5-Fluoro orotic acid (5-FOA), an analog of uracil, was used to evict the plasmid containing the URA3 gene. For FOA plate preparation, 1 mg 5’-FOA ml-1 was added after autoclaving the media. YPD was used for growth assay, and 1mM arsenous acid, 1mM sodium arsenate, 2mM copper sulphate, 2mM zinc sulphate, 1mM cadmium chloride and 1% lactic acid were added wherever required. The spot assay was done on the solid medium by applying dilutions of 104, 103, 102, and 101 colony forming unit (CFU) per spot.
2.2 Vectors, plasmid constructions and primers
The yeast vectors pRS315, pRS316 and pRS11326 were used for maintaining constructs and assessing the phenotypes. The plasmid pUC19 was used for deletion construct. The CCT7 gene (2153 bp) was amplified by PCR using primers KC124 and KC125 cloned into pJET1.2/blunt cloning vector. Subsequently, it was sub-cloned into pRS316 at BamHI-SalI site generating the plasmid pKA376. Upstream (591 bp) and downstream (506 bp) sequences of CCT7 were amplified using the primers KC126/KC127 and KC128/KC129. The PCR amplified products were cloned into vector pTZ57R/T. The primers KC120 and KC121 were used to amplify 1205 bp TRP1 gene and cloned into pJET1.2/blunt cloning vector. Plasmid pUC19 was used as a primary vector for plasmid constructs. 1.2 kb TRP1 gene was cloned in BamHI- SalI site, and 591bp BamHI-SacI upstream fragment was cloned in the BamHI-SacI site of pUC19. SphI-SalI 506 bp downstream fragment was cloned in the above construct in SphI-SalI site. The cassette was released by digesting with SacI and transformed into yeast strain B-8728 containing a copy of CCT7 in pRS316 plasmid. Gene deletion was done using the homologous gene disruption method. The primers KC316/ KC317 were used for internal amplification and KC542/KC129 were used for junction checking to verify the gene deletion. A point mutation was created from GGG to EGG of CCT7 using primers KC474/KC475 with CCT7 cloned in BamHI-SalI site of pRS315 using site-directed mutagenesis kit. For the aggregation visualization, the primers KC589/KC590 were used to amplify the HSP104 gene (3181 bp, promoter and 3’ end forming sequences) and cloned in BamHI-XhoI site of pRS11326. Subsequently, a DNA fragment (750bp) containing GFP was cloned at BamHI site to generate the plasmid pKA844. The GFP-tagged cassette was transformed in wild-type and mutant yeast (Sambrook and Russell 2001). The lithium acetate/ polyethylene glycol method was used to transform yeast cells (Hinnen et al. 1978). All the strains, plasmid constructs and primers used in the study are listed in Supplementary Table 1, 2, and 3.
2.3 Atomic Adsorption Spectroscopy:
Fresh yeast cells were inoculated in 20ml of YPD, grown till the early log phase. 1mM CdCl2was added to it and grown for 16 hours at 30oC, initial pH 6. Conditions were altered accordingly to study the effect of contact time and pH. The culture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was analyzed for residual cadmium by Perkin Elmer AAS, PinAAcle 900F. The Q, uptake capacity (µg mg-1) and E, removal efficiency (%) was calculated by
Where Ci (µg ml-1) is the initial concentration, Ce (µg ml-1) is the final concentration of cadmium, M (mg) is the dry weight of yeast, and V (ml) is the volume (Perez-Marin et al. 2007).
2.4 Microscopic methods
Strains were grown till the early-log phase then 1mM CdCl2 was added and grown for 4hours with and without cadmium. Cells were collected and washed with distilled water. Slides were prepared in 1% agar and seen under the Nikon ECLIPSE Ti-Efluorescent microscope. For electron microscopy, cells were spread over 1% agar, layered the coverslip, and desiccated. These were then sputtered with gold particles for 15s and imaged using scanning electron microscope Hitachi SU6600 (Japan). 5µl of 2mg neutral red ml-1 was added to 25µl of cell culture and incubated for 5 minutes to visualize vacuoles, and slides were seen under the optical microscope (Corbacho el al 2010).
2.5 RNA preparation and real-time PCR
Total RNA isolation was done using the hot acid-phenol method as described. Eppendorf NanoDrop spectrophotometer was used for estimation of RNA concentration. DNaseI treatment was given to remove any DNA contamination in RNA samples and verified with standard PCR. cDNA from RNA samples were prepared using Thermo Fisher Scientific RevertAid first strand cDNA synthesis kit. Real-time quantitative PCR was performed using Bio-Rad CFX96 Real-time system in 20µl reactions taking 25ng cDNA µl-1 in each sample using Thermo Fisher Scientific Maxima SYBR Green/ROX qPCR Master Mix. The in-built Bio-Rad system protocol (CFX_2 step ampl protocol) was used for RT-qPCR with additional melt curves after the amplification. Briefly, denaturation was done at 95oC for 3 minutes, followed by 40 cycles of 95oC for 10 seconds and 55oC for 30 seconds. After amplification, melt curves were run at 65oC to 95oC for 5 seconds at increment of 0.5oC. The gene expression difference was calculated as fold change using the ∆∆Ct method (Schmittgen and Livak 2008). Triplicates were performed in all the PCR reactions.
2.6 Bioinformatics methods and tools:
Peptide sequences of eight yeast CCTs were retrieved from the Saccharomyces Genome Database. Prime module (version 11.1) of Schrödinger, (Schrödinger, LLC, New York, NY, 2017) was used to predict three-dimensional structures. Template structures 5GW4_H was selected based on the percentage identity. Clustal omega was used for multiple sequence alignment of protein sequences. Web logo model for conserved motif was prepared using consurf (Ashkenazy et al. 2016; Sievers et al. 2011).