Yeast strains and growth conditions
The yeast strains used in this work included D. hansenii strain BCRC No. 21947, isolated from Hsilo County, Taiwan, S. cerevisiae Neo Type strain Y1 BCRC No. 21447 from brewer's top yeast, obtained from FIRDI (Food Industry Research and Development Institute, Hsin-chu City, Taiwan), and P. methanolica strain PMAD11 genotype ade2-11, obtained from Invitrogen, U.S.A. D. hansenii was cultured at 24°C in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% dextrose) while S. cerevisiae and P. methanolica were cultured at 28°C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) and YPAD medium (1% yeast extract, 2% peptone, 2% dextrose, 0.01% adenine), respectively.
RNA extraction and poly(A+) RNA purification
Total RNA was extracted with a modified hot phenol protocol . Poly (A+) RNA was isolated from total RNA using Mag-Net mRNA Isolation Kit according to the manufacturer's instruction (Amresco, Inc. USA). Concentration of RNA was determined using a NanoDrop spectrophotometer (NanoDrop, Wilmington, USA). RNA quality was verified by electrophoresis on 1.5% formaldehyde agarose gel and stained with ethidium bromide.
Subtractive hybridization and construction of subtracted cDNA library
Subtractive hybridization was performed using PCR-select cDNA Subtraction Kit (Clontech, Palo Alto, CA, U.S.A.). For screening of differentially upregulated genes, cDNA synthesized from the 2.5 M NaCl treated yeast cells for 24 min was used as the tester while that from non-treated cells served as the driver. The PCR products of forward subtraction were subcloned into the pGEMR-T Easy Vector (Promega, USA). Competent cells of E. coli (XL-Blue) was transfected with the plasmids and grown on LB-agar medium containing 5-bromo-4-chloro-3-indolyl-b-d-galactoside (X-gal) (Sigma, U.S.A.), isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma, U.S.A.) and ampicillin. Individual white colonies with insert DNA were randomly picked for further analysis.
Sequencing and sequence analysis
White clones from the forward subtractive hybridization libraries were sequenced with the universal T7 or SP6 sequencing primers using an automatic DNA sequencer (3100 Genetic Analyzer, ABI, U.S.A). All inserted sequences were queried for similarity through the NCBI database using BLASTX sequence comparison software http://www.ncbi.nlm.nih.gov/BLAST.
Quantification of DhAHPby quantitative real-time PCR (Q-RT-PCR)
Total RNA isolated from yeast cells treated with NaCl for various time intervals was first treated with DNase I (Promega, U.S.A.) to remove DNA contamination before cDNA synthesis . cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (P/N 4368814, ABI, U.S.A.) for RT-PCR according to the manufacturer's instruction. The sequence forward and reverse primers for Q-RT-PCR were designed using the primer ExpressR Software provided by Applied Biosystems. A set of D. hansenii 18S ribosomal RNA primers was designed for use as an endogenous control.
18S forward: G'-CGTCCCTGCCCTTTGTACAC-3'
18S reverse: G5'-GCCTCACTAAGCCATTCAATCG-3'
DhAHP target forward: G5'-GGAGCCCCAGGAGCATTTA-3'
DhAHP target reverse: G5'-TGGGCCAAATAATCGGGAAT-3'
Real-time PCR assay was carried out in an ABI PRISM 7500 Sequence Detection System (ABI, U.S.A.). The amplification of the target genes was monitored every cycle by SYBR-Green fluorescence.
Rapid amplification of cDNA ends (RACE)
The full-lengthed cDNA clone of DhAHP was obtained by rapid amplification of the cDNA ends using the GeneRacerTM Kit (Invitrogen, U.S.A.), as described in the manual provided by the manufacturer. The forward and reverse gene specific primers (GSPs) used for RACE were designed based on the DhAHP cDNA sequence. The universal primers for 5' and 3' Race were GeneRace 5' and GeneRace 3', respectively, provided in the kit. After PCR the DNA fragments were cloned into pGEMR-T Easy vector (Promega, U.S.A.) for sequencing.
Forward (GSP): 5'- GTCAATGCTGCTTGGGGTAAAGCTTTA-3'
Reverse (GSP):5'- GGTCTCAGCACTGGAAATTTCAGTG-3'
GeneRace 5':5'- CGACTGGAGCACGAGGACACTGA-3'
GeneRace 3':5'- GCTGTCAACGATACGCTACGTAACG-3'
The deduced amino acid sequence of DhAHP was analyzed with the Expert Protein Analysis System http://www.expasy.org/. Multiple sequence alignment was performed for sequence comparison and alignment of D. hansenii Ahp and two other reported AHPs (Swiss-Prot: P38013 and Q5AF44) from S. cerevisiae and C. albicans and peroxisomal membrane protein (Swiss-Prot: O14313) from S. pombe and three other structural homolog proteins (Swiss-Prot:Q8S3L0, B3GV28 and P30044) from P. tremula, P. sativum and H. sapiens. The alignment and phylogenetic analysis were carried out by the protein sequence alignment program CLUSTAL W.
Southern and northern hybridization analysis
Genomic DNA was isolated from yeast cells by the method of Hoffman and Winston . Southern and northern hybridization analyses were performed using the DIG High Prime DNA Labeling and Detection Starter Kit (Roche Diagnostics, Switzerland). For Southern hybridization, 20 μg genomic DNA was digested with EcoRI and BamHI and electrophoretically separated on 0.7% (w/v) agarose gels in TBE buffer and DNA fragments blotted onto nylon membrane (Amersham Pharmacia Biotech, U.K.) by 20×SSC. The full-lengthed DhAHP DNA was labeled and used as a hybridization probe. For nothern hybridization analysis, RNA was extracted from D. hansenii that was not treated or treated with 2.5 M NaCl for 16 min, separated by electrophoresis in formaldehyde gels, blotted onto nylon membrane and hybridized with labeled DhAHP DNA, as described above for Southern analysis.
Silencing of DhAHP expression in D. hanseniiby RNA interference
To test the function of DhAHP, RNA interference was employed to suppress its expression in D. hansenii using the Knockout RNAi System Kit (Clontech, U.S.A.), as described in the manual by the manufacturer. The oligonucleotide sequences including BamHI and EcoRI sites, target sense sequence, hairpin loop, target antisense sequence and terminator were shown as follow.
BamHI Target sense sequence Hairpin loop Target antisense sequence Terminator
A chemical method based on LiCl, as described by Tarutina and Tolstorukov , was used to transfect D. hansenii and the RNAi transformant was screened by its poor ability to grow on YM11 solid media containing 2.5 M NaCl. The transformant was confirmed by sequencing the introduced DNA fragment in the genome with specific primers and by Q-RT-PCR.
Overexpression of DhAHP in D. hansenii, S. cerevisiae and P. methanolica
To further test its functional role in relation to salt tolerance, DhAHP was overexpressed in three yeast species with contrasting degrees of salt tolerance. The entire ORF of DhAHP was first amplified by PCR utilizing the overexpression 5' primer, which introduced an EcoRI site in front of the starting ATG codon, and the overepression 3' primer, which introduced a BamHI site before the stop codon. This DNA fragment was inserted into the expression vector of pMETB (Invitrogen, U.S.A.). The plasmid DNA of the DhAHP/pMETB veector was digested with Pst I to release the P
/DhAHP expression cassette, which was then introduced into D. hansenii, S. cerevisiae and P. methanolica by a chemical method based on LiCl, as described by Tarutina and Tolstorukov . The AUG1 sequence is a methanol inducible promoter to drive the expression of introduced DhAHP. Functional complementation was used to screen transformants from the three species by culture on solid media containing 0.5% methanol and higher NaCl concentrations than they can normally tolerate. For isolation of D. hansenii overexpression transformants YM medium containing 3.5 M NaCl was used, for S. cerevisiae transformants YPD medium containing 1.5 M NaCl was used and for P. methanolica transformants YPAD medium containing 2.0 M NaCl was adopted. The transformants were confirmed by sequencing the P
DNA fragment in the genome with specific primers and by Q-RT-PCR with cells grown under high salt in the presence or absence of methanol. The ability of the selected transformants to tolerate salt was further assessed by growing in liquid media containing high NaCl concentrations.
Measurement of intracellular ROS
For measurement of cellular ROS, the redox-sensitive fluorescent probe 2', 7'-dihydrodichlorofluorescein diacetate (DCFA-DA) (Sigma, U.S.A.) was used according to Chattopadhyay et al. . The cells of wild type strains and DhAHP overexpression transformants were grown in appropriate liquid media without any salt for approximately 36 h (1 O.D. at 600 nm) and switched to fresh media containing high NaCl (3.5 M for D. hansenii, 2.0 M for S. cerevisiae and 2.5 M for P. methanolica) with or without methanol for 5 h. To determine ROS, cells were harvested by centrifugation and treated with 10 μM DCFA for 30 min at 30°C. The cells were re-suspended and washed in water and extracted by vortexing with glass beads. Extracts were centrifuged and fluorescence in the supernatant was measured with λEX = 485 nm and λEM = 524 nm in a fluorescence spectrophotometer (Infinite F200). Fluorescence signals were expressed relative to that of the wild type strain before any stress treatments (fold over control).