The food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown.
In this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance.
The transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.
The Saccharomyces cerevisiae transcription factor Haa1 was first identified based on the DNA binding domain (DBD) homology with the copper-regulated transcription factor Cup2 (alias Ace1) DBD . The paralogue pair Haa1 and Cup2 DBDs comprise 123 and 124 amino acid residues, respectively, at the N-terminal and include a conserved zinc module and a set of four cysteine-cysteine clusters organized in a consensus sequence that forms the copper regulatory domain (CuRD). Such conservation at the level of the DNA binding domains led to hypothesize that, like Cup2, Haa1 could play a role in copper homeostasis; however, metalloregulation and involvement of Haa1 in S. cerevisiae tolerance to copper could not be assigned to this transcription factor . Indeed, no physiological function could be ascribed to Haa1 until the description, by our laboratory, of the essential role of Haa1 in S. cerevisiae adaptation and tolerance to weak acids, especially to the short-chain hydrophilic acetic and propionic acids .
S. cerevisiae Haa1 is considered the main player in yeast genomic expression reprogramming in response to acetic acid stress, being involved in the direct, or indirect, transcriptional activation of approximately 80% of the acetic acid-responsive genes, several of which required for maximum tolerance to this weak acid [3, 4]. The Haa1 target genes are involved in transcription, multidrug resistance, cell wall remodelling, metabolism of lipids, carbohydrates and amino acids, and nucleic acid processing [2, 4]. Haa1 binds, in vivo, to an acetic acid responsive element (ACRE) in the promoter of its target genes . Among these genes are TPO2 and TPO3 that code for two plasma membrane transporters of the Major Facilitator Superfamily proposed to mediate the efflux of acetate from the cell interior in acetic acid challenged yeast cells [2, 4, 5]. Other genes of the Haa1 regulon that are required for tolerance to acetic acid [1, 4], include: YGP1 (cell wall-related secretory glycoprotein, ), YRO2 (plasma membrane protein with a putative role in acetic acid tolerance ), HRK1 (a protein kinase of a family related with the phosphorylation of membrane proteins and implicated in activation of the activity of plasma membrane H+-ATPase Pma1 ) and HSP30 (a plasma membrane heat shock protein proposed as a negative regulator of Pma1 ). The involvement of Haa1 in S. cerevisiae adaptation and tolerance to acetic acid stress has been demonstrated [2, 4, 5], but the function of Zygosaccharomyces bailii Haa1 homologue remains unknown. However, this yeast species is highly problematic in the spoilage of acidic food and beverages due to its remarkable capacity to tolerate acetic acid and other weak acid food preservatives . Although the mechanisms underlying the response and extreme tolerance of Z. bailii to acetic acid are still poorly characterized, a number of relevant physiological strategies have been reported. These include the capacity of the yeast cells to tolerate short-term intracellular pH changes [11, 12], co-consume acetic acid and glucose [13–15] and exhibit high basal level of complex sphingolipids proposed to decrease plasma membrane permeability to this weak acid . Also, a recent genome-wide study identified the Z. bailii transcription factor ZbMsn4 , homologous to the S. cerevisiae stress-responsive transcriptional activators Msn4 and Msn2 , as an acetic acid tolerance determinant. Other tolerance determinant genes, homologous to S. cerevisiae GYP8 and WSC4 (cellular transport and transport routes), PMT1, KTR7 and RKR1 (protein fate), TIF3 (protein synthesis) and ILV3 (amino acid metabolism) were also singled out in the same study . In S. cerevisiae, MSN2 was found to be an acetic acid tolerance determinant , and MSN4 is among the genes activated by Haa1 in response to acetic acid stress .
Based on the amino acid sequence similarity of Haa1 and Cup2 DBDs, Keller et al.  proposed that HAA1 and CUP2 are paralogues. The concept that this paralogy relationship originated in the whole genome duplication (WGD) event was first proposed by Dietrich and co-authors , and independently confirmed upon the release of the Yeast Gene Order Browser (YGOB) [20, 21], a database dedicated to the assignment of an orthology/ohnology classification to WGD-originated genes in the Saccharomycetaceae family. Although the identification of the orthologue/ohnologue status is important to trace the evolutionary history of a particular gene family, there are other forces driving the evolution of genes and genomes, as for example local duplications, horizontal gene transfer, gene loss and gain and gene conversion. Understanding the origin of those variations requires the identification of the complete set of bona fide members of a gene family and the integration of different in silico approaches, such as phylogeny and gene neighbourhood analysis.
In the present study we investigate the function of Z. bailii gene ZbHAA1 using functional and evolutionary approaches. Using a blastp network traversal approach to perform a global analysis of all the ORFs encoded in the publicly available genomes of 28 Saccharomycetaceae yeast species, a group of amino acid sequences was identified as showing strong sequence similarity to S. cerevisiae Haa1 amino acid sequence. The function of the sole ZbHAA1 encoded in the genome sequences of Z. bailii strains CLIB 213T (mentioned herein as ZYBA_3_9_I00670)  and IST302 (ORF ZBIST_2620) (Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished) was examined. Z. bailii strain IST302 has recently been used by our laboratory because, contrasting with the hybrid strain ISA1307 and Z. bailii CLIB 213T, it is more prone to genetic engineering and physiological studies (, Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished). Phylogenetic and gene neighbourhood analyses of the amino acid sequences of the Haa1 homologues identified in Saccharomycetaceae yeasts, led us to confirm that, upon the WGD event , the single transcription factor encoded in the protoploid ancestor yeast species has evolved into two distinct gene sub-lineages, each corresponding to the homologues of S. cerevisiae HAA1 and CUP2 genes in post-WGD species. In this work we experimentally tested the possibility that both HAA1 and CUP2 genes have arisen from the subfunctionalization of the ancestral HAA1/CUP2 orthologue after the WGD event.
Strains and growth media
The prototrophic strains Zygosaccharomyces bailii IST302  and the type strain Z. bailii ATCC58445T (=CLIB 213T)  were used in this work. Auxotrophic strains Saccharomyces cerevisiae BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and derived deletion mutants haa1Δ and cup2Δ were obtained from the EUROSCARF collection. Prototrophic yeast strains were batch-cultured at 30 °C with orbital agitation (250 rpm) in liquid mineral medium (MM) that contains, per litre, 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4 (Difco), 20 g D-glucose (Merck) and 2.65 g (NH4)2SO4 (Merck). S. cerevisiae auxotrophic strains were cultured in MM4 medium, prepared using MM medium supplemented with 20 mg methionine, 20 mg histidine, 60 mg leucine and 20 mg uracil (all from Sigma). Yeast cells used for yeast transformation experiments were cultured in YPD medium (1% yeast extract, Difco, 2% peptone, Difco, and 2% D-glucose, Merck). For Z. bailii transformants recovery, cells were grown in YPF medium (2% D-fructose, Amresco, 1% yeast extract, Difco, and 2% peptone, Difco). Transformant selection after disruption of ZbHAA1 in the chromosome, or after transformation of Z. bailii IST302 with an extra copy of ZbHAA1, was performed in plates containing YPF supplemented with 200 μg/mL G418. Selection of transformants from the derived auxotrophic S. cerevisiae strains was based on growth in MM4 without uracil supplementation. Escherichia coli XL1-Blue was used for plasmid maintenance and general cloning procedures. E. coli cells were grown in Luria-Bertani medium (LB), supplemented with 150 mg/L ampicillin when required. Solid media were obtained by adding 20 g of agar to each litre of the corresponding liquid media. All strains were maintained at −80 °C in appropriate media supplemented with 15% glycerol (v/v).
ZbHAA1 and ScHAA1 cloning into pGREG506
The nucleotide sequences of ZbHAA1 from Z. bailii IST302 (ZBIST_2620) and CLIB 213T (ZYBA_3_9_I00670) were obtained from the corresponding genome sequences (Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished, and Galeote et al. , respectively). The pGREG506 vector from the DRAG & DROP collection  was used to individually clone by homologous recombination and expression of the ORFs ZBIST_2620, ZYBA_3_9_I00670 and ScHAA1, all under the control of the same promoter region, the ScHAA1 promoter region. The cloning vector pGREG506 was acquired from EUROSCARF and contains a HIS3 gene under the control of a galactose inducible promoter (GAL1) and the yeast selectable marker URA3. The homologous recombination was performed in two steps comprising, first, the cloning of ScHAA1 promoter into pGREG506 and, second, the cloning of ScHAA1 or ZbHAA1 into the recombinant vector obtained during the first step. ScHAA1 promoter was amplified from strain BY4741 using the primers ScHAA1prom-F and ScHAA1prom-R (Additional file 1). The amplified ScHAA1 promoter and pGREG506 digested with SpeI and SacI restriction enzymes, i.e. pGREG506 without the GAL1 promoter, were co-transformed into S. cerevisiae BY4741 parental strain and a recombinant plasmid (pGREGpromScHAA1) was obtained through homologous recombination in yeast. Correct cloning of the promoter was confirmed by DNA sequencing. The ORFs ZBIST_2620, ZYBA_3_9_I00670 and ScHAA1 were amplified from Z. bailii IST302, Z. bailii CLIB 213T and S. cerevisiae BY4741 genomic DNA, respectively, using the corresponding pair of primers ZbHAA1rec-F/ZbHAA1rec-R and ScHAA1rec-F/ScHAA1rec-R (Additional file 1). Each of the amplified ORFs was co-transformed into S. cerevisiae BY4741 with the recombinant vector pGREGpromScHAA1 linearized with SalI (i.e. with the HIS3 gene removed). Correct cloning of the genes into the recombinant plasmids was confirmed by DNA sequencing. These plasmids, named here as pG_ScHAA1, pG_ZbHAA1-IST and pG_ZbHAA1-CL, containing, respectively, ScHAA1 or ZbHAA1 homologue from strain IST302 or strain CLIB 213T, were then transformed into S. cerevisiae haa1Δ deletion mutant. pG_ScHAA1 and pG_ZbHAA1-IST were also transformed in cup2∆ deletion mutant. Additionally, S. cerevisiae BY4741 parental, haa1Δ and cup2∆ were transformed with the cloning vector pGREG506 with the HIS3 gene deleted (pGREG506_noHIS3) that was previously constructed and reported as the correct empty vector to be used as control  since in the recombinant vectors the HIS3 gene is substituted by the insert of interest.
Disruption of Z. bailii IST302 HAA1 homologue
Construction of ZbHAA1 disruption cassette
The disruption of Z. bailii IST302 ZbHAA1 (ORF ZBIST_2620) was performed by homologous recombination of a ZbHAA1 disruption cassette into the chromosome. The disruption cassette was obtained by a three step-based fusion of DNA fragments generated by polymerase chain reaction (PCR) [25, 26]. All PCR reactions were performed with Taq Phusion High-Fidelity DNA Polymerase (Thermo Scientific) following the manufacturer’s instructions. In the first step three primary PCR reactions were carried out. The amplification of fragments haa1a and haa1b was obtained by using the pair of primers haa1a-1/haa1a-2 and haa1b-3/haa1b-4, respectively, with Z. bailii IST302 genomic DNA as a template. The kan cassette DNA fragment was amplified from the centromeric plasmid pZ3bT  using the pair of primers kan-5/kan-6 (Additional file 1). These primers were carefully designed in order to have identical melting temperatures. Accordingly, the same annealing temperature (56 °C) was used in all PCR reactions. The amplified DNA fragments haa1a, haa1b and kan cassette were analysed in a 0.8% agarose (NZYTech) gel electrophoresis and its molecular weight confirmed by comparison with 1 kb Plus DNA Ladder (Invitrogen). The DNA fragments were then directly extracted and purified from the agarose gel using JETQUICK gel extraction spin kit (Genomed) according to manufacturer’s instructions. In the second and third steps we followed the methodology described by Shevchuk et al.  with minor modifications. Basically, a PCR reaction of 12 cycles was carried out without primers and containing approximately 100 ng of haa1a, 100 ng of haa1b and 30 ng of kan cassette. Subsequently, a total of 3 μL of this PCR product was used as a template for another PCR reaction (total volume of 20 μL) containing the primers haa1a-1 and haa1b-4 (Additional file 1). The resulting 2.7 kb PCR product was analysed by electrophoresis in 0.8% agarose, extracted and purified as mentioned above. The methodology was performed several times to obtain 0.5–1 μg of DNA that was subsequently used for transformation of Z. bailii IST302 cells.
Transformation of Z. bailii IST302 and homologous recombination of ZbHAA1 disruption cassette in the chromosome
The transformation of Z. bailii IST302 was performed with Alkali-CationTM Yeast Transformation kit (MP Biomedicals) introducing minor modifications to the manufacturer’s protocol. Specifically, cells were cultivated overnight in YPD medium, at 30 °C and 250 rpm, and then re-inoculated at an absorbance of 0.1 at 600 nm in 50 ml of fresh medium. Cells were grown until mid-exponential phase (absorbance of 1.0 ± 0.1 at 600 nm) and a total of 1.5x108 cells were collected by centrifugation. All centrifugation steps were carried out during 10 min at 4000 rpm (4 °C). A total of 0.5–1 μg of the HAA1 disruption cassette was added to the transformation mixture. The transformation reaction was incubated during 30 min at 30 °C, followed by a heat-shock at 42 °C during 40 min. Transformed cells were then centrifuged, resuspended in YPF containing 1 M sorbitol (Sigma) and incubated overnight at 30 °C for recovery. Yeast recombinant clones were selected in YPF plates containing 200 μg/mL G418 (Sigma).
Cloning and expression of an extra copy of ZbHAA1 in Z. bailii IST302
The expression of an extra copy of ZbHAA1 gene in the parental strain Z. bailii IST302 was performed by cloning this ORF by homologous recombination into the centromeric expression vector pZ3bT  linearized with XbaI. The amplification of ZbHAA1 and of its corresponding promoter (approximately 1000 bp upstream the start codon) from Z. bailii IST302 genomic DNA was performed using the primers ZbHAA1pZ-F, ZbHAA1pZ-R (Additional file 1). The amplified product was co-transformed with the linearized pZ3bT into S. cerevisiae BY4741. Selection of the transformants harbouring the recombinant vector pZ3bT_ ZbHAA1 was performed in YPD plates containing G418 (200 μg/mL). The correct recombination was confirmed by sequencing the obtained plasmids. The recombinant plasmids and the cloning plasmid were used to transform Z. bailii IST302, as previously described.
Susceptibility assays in liquid media
Susceptibility assays in liquid media were assessed by comparing yeast growth curves obtained by periodic measurements of culture absorbance at 600 nm. Yeast cells were pre-cultured in the appropriate unsupplemented medium until mid-exponential phase (standard absorbance of 0.5 ± 0.05) and then reinoculated at an initial absorbance of 0.05, in 50 ml of the fresh medium, supplemented or not with acetic acid. All assays were carried at 30 °C with orbital agitation (250 rpm). Different growth media were used according to strains’ nutritional requirements. The susceptibility to acetic acid of S. cerevisiae parental strain BY4741 and the derived deletion mutant haa1Δ, harbouring each of the recombinant plasmids produced in this study was assessed in liquid MM4-U medium, pH 4.0, either or not supplemented with 60 mM or 75 mM acetic acid. Acetic acid susceptibility assays involving Z. bailii strains were performed in liquid MM medium, pH 4.0, either or not supplemented with 180 and 220 mM acetic acid.
Susceptibility assays in solid media
Yeast cells were pre-cultured overnight in the appropriate unsupplemented liquid medium and then reinoculated at an absorbance at 600 nm of 0.05, in 50 ml of fresh medium. When the cultures reached an absorbance of 0.5 ± 0.05 at 600 nm cells were resuspended in sterile water to obtain suspensions with an absorbance of 0.05 ± 0.005. These cell suspensions and three subsequent dilutions (1:5; 1:10; 1:20) were applied as 4 μL spots onto the surface of agarized media, supplemented, or not, with increasing concentrations of the compound to be tested and incubated at 30 °C for 3 to 5 days. In the case of Z. bailii IST302 parental and derived Zbhaa1∆ deletion mutant strains, as well as Z. bailii IST302 expressing an extra copy of ZbHAA1 (pZ3bT_ZbHAA1) or the cloning vector (pZ3bT) susceptibility to acetic, benzoic and sorbic acids and copper was compared in MM medium, at pH 4.5. Regarding S. cerevisiae BY4741 parental and derived deletion mutants haa1∆ and cup2∆ or this cup2∆ mutant transformed with the recombinant plasmids produced herein, susceptibility assays were assessed in MM4 or MM4-U media, respectively, at pH 4.5.
Comparison of mRNA relative levels by real time RT-PCR
The effect of the expression of ZbHAA1 in the mRNA levels from S. cerevisiae Haa1- or Cup2- target genes under acetic acid or copper stresses, respectively, was assessed by expressing the recombinant plasmids pG_ZbHAA1-IST, pG_ScHAA1 and the cloning vector either in S. cerevisiae haa1∆ or cup2∆ deletion mutants. S. cerevisiae BY4741 parental strain transformed with the empty vector was used as a positive control. The effect of ZbHAA1 in Z. bailii IST302 transcriptional response to acetic acid was also assessed by comparing the mRNA levels from Z. bailii genes homologous to S. cerevisiae Haa1-regulon genes in cells of Z. bailii IST302 parental and derived deletion mutant Zbhaa1∆ strains, in the presence and absence of appropriate concentrations of acetic acid. In both assays, yeast cells were cultivated in the appropriate media until mid-exponential phase (standard absorbance at 600 nm of 0.6 ± 0.05) and then reinoculated (initial absorbance of 0.2 ± 0.01) in 500 mL unsupplemented media. After 1 h of growth 100 mL of cell culture were collected and set as the control condition (Time point 0); simultaneously, acetic acid or copper were added to the remaining 400 mL of cell culture. A total of 100 mL of acetic acid- or copper-stressed cells were harvested 1 and 2 h after the addition of acetic acid or copper (Time points 1 and 2). Cells were collected by centrifugation (8000 rpm, 10 min) at 4 °C, washed twice with cold water and the pellets were frozen in liquid nitrogen and kept at −80 °C until RNA extraction. Gene transcript levels were assessed by real time RT-PCR. Extraction of total RNA was performed using a modified hot phenol method . Purification of RNA and DNA digestion with DNAse was performed using the commercial kit Nucleospin ® RNA (Macherey-Nagel, Germany). Synthesis of cDNA from total RNA samples was performed using the MultiscribeTM reverse transcriptase kit (Applied Biosystems) and the subsequent Real Time PCR step was carried out using SYBR_Green reagents and 7500 Real Time PCR System (Applied Biosystems). Specific primers were designed in Primer Express Software (Applied Biosystems) using gene sequences obtained from Z. bailii IST302 genome sequence (Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished) (Additional file 2) or S. cerevisiae genome database (http://www.yeastgenome.org/). The primer sequences are listed in Additional file 1. Relative values obtained for the expression of each gene either in Z. bailii IST302 parental strain or in S. cerevisiae BY4741 transformed with the cloning vector cultivated in control conditions (Time point 0) were set as 1 and the remaining values are presented relative to that sample. Relative gene expression between the conditions tested was calculated according to the 2-∆∆Ctmethod  using either ACT1 or ZbACT1 (in the case of S. cerevisiae or Z. bailii strains, respectively) as an internal control for input cDNA normalization. Experiments were performed, at least, three times. Results were analysed by two-way ANOVA, considering a p-value below 0.05, when comparing the relative mRNA levels in each strain at a given time point.
Identification of the putative Haa1/Cup2 homologues encoded in the genomes of the Saccharomycetaceae yeast species
The translated ORFs of the 33 sequenced hemiascomycetous yeast strains analysed in this work (Additional file 3) were retrieved from their corresponding genome databases (Table 1) and were compiled in the in-house Genome DB. These 33 hemiascomycetous strains correspond to 28 different species, all taxonomically classified in the Saccharomycetaceae family, in particular the genome sequences of Z. bailii CLIB 213T and Z. bailii IST302 (to be soon released) . Henceforth, the four letters code shown in Table 1 for species abbreviation will be used to designate both yeast genes and species. The number displayed after the first four letters is used to abbreviate the strain name when the genome of more than one strain from a given species is available. To standardize the annotation used, translated ORFs are represented with small letters. The comparative genomics approach used in this study is based on the sequence clustering of all translated ORFs of the 33 sequenced yeast strains. This required the compilation and organization of a total of 199443 translated ORFs. These translated ORFs were organized into a blast database and compared all-against-all using blastp algorithm made available in blast2 package . The blastp algorithm used a gapped alignment with the following parameters: open gap (−1), extend gap (−1), threshold for extending hits (11) and word size (3). This approach generated a total of 45.5 million pairwise alignments. In order to handle this amount of data, sequence clustering was formulated as a graph traversal problem, where the nodes are the translated ORFs and the edges indicate the existence of pairwise sequence similarity between amino acid sequences. Classification of the translated ORFs into clusters was achieved by breadth-first traversing this network at different e-value thresholds, ranging from E-60 to E-1. The identification of the putative Haa1/Cup2 homologues was achieved by traversing these blastp sub-networks using as starting node the S. cerevisiae Haa1 protein.
Phylogenetic analysis and tree construction methods
The amino acid sequences of the gathered translated ORFs were used to build a multiple alignment using MUSCLE software . This multiple alignment was analysed using the Jalview software  and processed using the MrBayes [34, 35], a Bayesian Markov chain Monte Carlo (MCMC) package for phylogenetic analysis. The functions made available in the seqinr and ape R packages  were used to convert the multiple alignment in fasta format into a nexus file required as input by MrBayes. Metropolis coupling of the MCMC sampling of the target distribution made available by the MPI (Message Passing Interface) version of MrBayes was used to speed up phylogeny computation. MrBayes MPI was set to use one “cold” chain together with 9 heated chains, while the remaining MPI parameters were set to default values. The MCMC simulations used 1200000 generations and two independent runs (each started from two distinct random trees) to confirm parameter convergence of the posterior probability distribution and, hence, assuring convergence into similar phylogenetic trees. The option of estimating the fixed-rate amino acid model made available by MrBayes was used, allowing the MCMC sampler to explore all of the nine available models by regularly proposing new ones (upon parameter convergence, each model contributes to the results in proportion to its posterior probability) and rate variation over sites was assumed to follow a gamma distribution. The maximum likelihood PROTML algorithm made available by the PHYLIP package coupled with bootstrap sampling  was used to confirm the results obtained with the MrBayes software suite (Additional file 4). The Dendroscope software was used for tree visualization .
Gene neighbourhood analysis
The package “sqldf”  and complementing scripting in R language was used to retrieve fifteen neighbour genes on each side of the query genes as well as the corresponding sequence clustering classification from Genome DB. The rationale of synteny analysis was described before [40, 41].
The existence of synteny between query genes was verified through the analysis of network topology (number of shared neighbour pairs) and the biological information associated with the corresponding edges. Three sources of biological information were used to assess the strength of each neighbour pair connection : i) closeness of the connecting neighbours in relation to the query genes, ii) sequence similarity between connecting neighbours and iii) dimension of the sequence cluster to which the homologous neighbours belong; small dimension of the sequence cluster indicates that it is small the probability that two homologous neighbours are in the vicinity of two query genes by chance.
Identification of S. cerevisiae Haa1 homologues in Z. bailii and in other Saccharomycetaceae yeast species
The Haa1 protein from S. cerevisiae S288c was selected as the starting node for the traversal of the blastp network. The iterative constraining and traversing of this pairwise similarity network at different e-values allowed the identification of the homologues of Haa1 transcription factor encoded in the genome sequences of Z. bailii CLIB 213T and IST302 and in other 31 Saccharomycetaceae strains (corresponding to a total of 28 species) (Table 1). The plot representing the number of sequences retrieved at different e-values shows four distinct blastp clustering ranges (Fig. 1). The first range occurs between e-values E-60 to E-40, gathering amino acid sequences highly similar to the starting node Haa1. In the second blastp clustering range (e-values from E-30 to E-13), the amino acid sequences similar to S. cerevisiae Cup2 protein are merged with the Haa1 protein set into a single group comprising a total of 51 sequences. In the third blastp clustering range (e-values from E-12 to E-2), the homologues of the S. cerevisiae Mac1 transcription factor are merged with the Haa1/Cup2 protein set (a total of 87 amino acid sequences). In the fourth blastp clustering range (e-values bellow E-1) many false positive amino acid sequences were incorporated in the Haa1/Cup2/Mac1 protein set. The cause for the merge of the Mac1 protein set with the Haa1/Cup2 homologues at low blastp e-values is the sharing of copper fist domain in the N-terminal of all these S. cerevisiae transcription factors. Considering that Mac1 is remotely similar to the Haa1 and Cup2 proteins, an e-value of E-13 was selected to constrain the pairwise similarity network and the corresponding 51 amino acid sequences were retained for further analysis (Additional file 3). The joint retrieval from the blastp network of both S. cerevisiae Haa1 and Cup2 transcription factors support the previous suggestion that the encoding genes constitute a paralogue pair [1, 19, 20] and allowed to conclude that the genome sequences of the two Z. bailii strains scrutinized in this study encode a single Haa1 homologue (Table 1) that is also homologous to Cup2. Consistent with the joint retrieval of Haa1 and Cup2 homologues, that in S. cerevisiae correspond to two independent transcription factors with 694 and 225 amino acids, respectively, the amino acid sequences herein identified in 28 Saccharomycetaceae species comprise highly heterogeneous protein sizes. Similar to the S. cerevisiae Haa1 (ScHaa1), Z. bailii sole Haa1/Cup2 homologue (ZbHaa1) has a protein size of 694 amino acid residues.
Expression of the HAA1 homologue from Z. bailii leads to increased tolerance of S. cerevisiae to acetic acid
To investigate the suggested role of the HAA1 homologues from two Z. bailii strains in acetic acid tolerance, the susceptibility to acetic acid of S. cerevisiae BY4741 and derived deletion mutant haa1Δ strains expressing either ZbHAA1 or ScHAA1 was compared (Fig. 2). Two ZbHAA1 gene alleles were considered in this analysis, one from Z. bailii IST302 (ORF ZBIST_2620, Additional file 2 (Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished) and the other from Z. bailii CLIB 213T . Both nucleotide sequences share 99.7% identity, with the corresponding encoded proteins having three amino acid substitutions. These ZbHAA1 genes were cloned into pGREG506 under the control of S. cerevisiae BY4741 HAA1 promoter. S. cerevisiae BY4741 HAA1 gene sequence was also cloned into the same vector, under the control of its native promoter and used as an internal control for comparative expression experiments. The expression of either ScHAA1 or the two ZbHAA1 genes in S. cerevisiae BY4741, cultivated in supplemented media with 60 or 75 mM acetic acid, significantly reduced the duration of the latency phases and increased the specific growth rates when compared with the host strain harbouring the cloning vector (Fig. 2a, Table 2). Interestingly, the increase of acetic acid tolerance of S. cerevisiae parental strain was maximal when ZbHAA1 from the more tolerant yeast strain Z. bailii IST302 (Palma M et al.: Genome sequence of the highly weak-acid-tolerant Zygosaccharomyces bailii IST302, amenable to genetic and physiological manipulations, unpublished) was expressed (Table 2). Also, the heterologous expression of ZbHAA1 gene alleles in haa1Δ mutant strain was found to rescue the susceptibility phenotype of the deletion mutant by increasing its tolerance to 60 mM acetic acid up to the level of the haa1Δ mutant expressing ScHAA1 gene (the duration of the latency phase is reduced of approximately 30 h) (Fig. 2b; Table 2). Differences registered in acetic acid tolerance in the parental strain harbouring the cloning vector and in the haa1∆ mutant expressing ScHAA1 (Fig. 2; Table 2) are likely the result of ScHAA1 expression from a centromeric vector  and not from the chromosomal locus. In fact, the mRNA levels from ScHAA1 in haa1∆ expressing ScHAA1 from the recombinant vector are above those in the parental strain transformed with the cloning vector, both in the absence (Time point 0) or presence of acetic acid stress (Time points 1 and 2) (Fig. 3a). However, the mRNA levels from ZbHAA1 in haa1∆ expressing ZbHAA1 could not be calculated because the parental strain used as a reference strain for the calculation of relative mRNA levels does not harbour the ZbHAA1 gene (Fig. 3b).
ZbHaa1 is involved in the regulation of S. cerevisiae acetic acid-responsive gene YGP1
The mRNA levels from YGP1 gene in S. cerevisiae, whose transcriptional activation under acetic acid stress is dependent on Haa1 , were compared in S. cerevisiae haa1Δ mutant expressing either ScHAA1 or ZbHAA1 and in the parental strain and derived haa1Δ mutant transformed with the cloning vector (Fig. 3c). As expected, the relative mRNA levels from YGP1 gene in the haa1∆ mutant were below the levels in the parental strain, both in the absence of acetic acid (Time point 0) or following sudden exposure to acetic acid (Time points 1 and 2). An increase in mRNA levels from YGP1 gene was detected in the haa1Δ mutant expressing ZbHAA1 or ScHAA1, respectively after 1 and 2 h of acetic acid supplementation of the growth medium, but no activation was detected in haa1∆ harbouring the cloning vector (Fig. 3c). These results indicate that ZbHaa1 is also able to mediate the transcriptional activation of YGP1 in S. cerevisiae cells challenged with acetic acid stress.
ZbHAA1 is required for Z. bailii tolerance to several weak acids
To examine the hypothesized role of the expression of ZbHAA1 in Z. bailii tolerance to acetic acid, a Zbhaa1∆ deletion mutant was prepared in Z. bailii IST302 and the susceptibility of the parental strain and derived mutant to acetic acid, as well as to other weak acids used as food preservatives (benzoic and sorbic acids), was compared by spot assays (Fig. 4a). Using equivalent concentrations of the different weak acids or, in other words, concentrations that induce a similar duration of growth latency of the parental strain, it was possible to demonstrate that ZbHAA1 expression is required for increased Z. bailii tolerance to the three weak acid food preservatives tested, but ZbHAA1 protective effect was more evident under acetic acid stress (Fig. 4a). In fact, no cell growth was detected in the Zbhaa1∆ mutant strain after 4 days of incubation with 220 mM acetic acid, whereas this mutant strain was still able to resume growth after a latency of approximately 4 days when cultivated in the presence of equivalent concentrations of the more lipophilic benzoic and sorbic acids. This result is in line with results reported before for the effect of HAA1 in S. cerevisiae indicating that maximal protection is exerted against the short-chain length more hydrophilic weak acids .
Weak acid concentrations that impaired growth of Z. bailii IST302 harbouring the cloning vector were used to compare the tolerance of Z. bailii IST302 expressing an extra copy of ZbHAA1 (Fig. 4b) that led to a remarkable increase of Z. bailii tolerance to the three weak acids (Fig. 4b). The enhanced tolerance to acetic acid of Z. bailii IST302 expressing an extra copy of ZbHAA1 was also confirmed in liquid MM medium supplemented with 220 mM acetic acid at pH 4.0, where a decreased duration of the latency phase of approximately 30 h was observed (Fig. 4c). In summary, ZbHAA1 is an important player in Z. bailii tolerance to acetic acid and other weak acid food preservatives.
ZbHaa1 is required for acetic acid-induced transcriptional activation of Z. bailii genes homologous to S. cerevisiae Haa1-target genes
The effect of the presence of acetic acid stress and the expression of ZbHAA1 in the transcription levels from six Z. bailii genes homologous to S. cerevisiae genes whose transcription was previously reported to be dependent on Haa1 regulation under acetic acid stress , was examined (Fig. 5). The nucleotide sequences of the referred Z. bailii genes, homologous to S. cerevisiae Haa1 target genes, specifically ZbHRK1, ZbHSP30, ZbTPO3, ZbMSN4, ZbYGP1 and ZbYRO2, are available in Additional file 2. The mRNA levels from the selected putative ZbHaa1 targets in Z. bailii IST302 were found to increase during the early adaptive response to the acid, specifically 1 h after sudden exposure to acetic acid, this activation being significantly reduced in Zbhaa1∆ deletion mutant (Fig. 5). These results indicate that the activation of the selected genes in response to acetic acid stress is dependent on ZbHaa1, and suggest that the Haa1 regulon is similar in S. cerevisiae and Z. bailii species.
Phylogenetic analysis of the Haa1 and Cup2 homologues in Saccharomycetaceae yeasts
Using the multiple alignment of the amino acid sequences of the putative Haa1 and Cup2 homologues of 33 strains from 28 pre- and post- WGD species, including Z. bailii CLIB 213T and IST302 sequences, as input, MrBayes phylogenetic software suite was used to construct a phylogenetic tree representing this family of transcription factors with copper-fist binding domain. After running two MCMC simulations for 1200000 generations, the standard deviation of split frequencies was below 0.01 and the potential scale reduction factor (PSRF) was reasonably close to 1.0 for all parameters, indicating their convergence [42, 43]. The parameters of the Gamma distribution, assumed being equal by MrBayes, converged into a value of 1.145, with the 95% Highest Posterior Density (HPD) interval ranging from 1.037 to 1.262, while the total tree length of the phylogenetic tree converged into a value of 37.112 (95% HPD interval = 34.139–40.085). The corresponding consensus Bayesian phylogenetic tree was retained for further analysis. A maximum likelihood (ML) approach made available by the PhyML software suite was used to confirm the phylogenetic relationships between the Haa1 and Cup2 homologues. The credibility of the phylogenetic clades was confirmed by inspection of the bipartition probabilities and bootstrapping values calculated for each internal node of the Bayesian and ML trees, respectively. Since the Bayesian and the ML statistical approaches originated similar phylogenetic trees regarding cluster composition and credibility (Fig. 6, Additional file 4), the analysis of the phylogenetic relationships between these transcription factors made in this study is based only in the tree obtained with MrBayes.
The analysis of the multiple alignment of the Haa1 and Cup2 transcription factor homologues revealed that their transactivation domain is poorly conserved in the Saccharomycetaceae yeast species under study, being responsible for the long branches observed in the corresponding tree (Fig. 6). This phylogenetic tree is divided into eight clusters, labelled from A to H. The translated ORF teph_1_d00690 was chosen as outgroup of this phylogenetic tree (cluster A) due to the strong dissimilarity of its amino acid sequence when compared with the ones of the remaining members of this gene family. The division of the Haa1 and Cup2 homologues into the proposed phylogenetic clusters was based on the following criteria: 1) protein length and 2) the taxonomic classification of the yeast species encoding each putative transcription factor. Cluster H comprises only proteins ranging from 221 to 356 amino acids (260 amino acids in average) and the remaining clusters comprise proteins ranging from 525 to 893 amino acids (671 amino acids in average). Nevertheless, the analysis of the amino acid and nucleotide sequences confirmed that all the phylogenetic clusters comprise full-size proteins, being free of fragments, pseudogenes and other type of sequence artefacts. The protein size and the analysis of the amino acid sequence similarity relationships shown by these transcription factors suggests that cluster H comprises the orthologues of S. cerevisiae Cup2 and clusters A, B, C, D and G comprise the orthologues of S. cerevisiae Haa1. With the exception of Tetrapisispora blattae, the genomes of the yeast species that have diverged after the WGD event encode two proteins belonging to this gene family, each one corresponding to the Haa1 or the Cup2 orthologues. Reinforcing the notion that these two S. cerevisiae genes are paralogues with origin in the WGD event [1, 19, 20] is the fact that the genomes of the protoploid Saccharomycetaceae species analysed in this study encode a single Haa1/Cup2 homologue (clusters E and F). Consistently, the genomes of the yeast species classified in the Zygosaccharomyces taxonomic genus under analysis in this study encode a single Haa1/Cup2 homologue: Z. bailii IST302 (ZBIST_2620), Z. bailii CLIB 213T (ZYBA_3_9_I00670) and Z. rouxii CBS 732 (ZYRO_1_F04862g).
All-against-all Needleman-Wunsch alignments were constructed to cross-compare the amino acid sequence similarity between the possible combinations of these three groups of proteins: Haa1 orthologues (clusters A, B, C, D and G), Cup2 orthologues (cluster H) and Haa1/Cup2 orthologues encoded in the protoploid Saccharomycetaceae genomes (clusters E and F). The amino acid sequence of the Haa1 and Cup2 orthologues showed, in average, 29.7% and 15.0% identity with Haa1/Cup2 protoploid orthologues, respectively (with the pairwise alignment comprising 63.5% and 31.7% of gaps, respectively). On the other hand, the comparison between the Haa1 orthologues versus the Cup2 orthologues showed that these share, in average, 12.7% identity between the corresponding amino acid sequences (and 67.4% of gaps). Considering that the protein size of the post-WGD Cup2 orthologues is less than one half of the size shown by the post-WGD Haa1 orthologues and of the protoploid Haa1/Cup2 orthologues, it is likely that the lower sequence identity between Cup2 orthologues and the members of other phylogenetic clusters is essentially due to the heterogeneous size and gap introduction in the pairwise sequence alignments.
Reconstruction of the evolutionary history of Haa1 and Cup2 orthologues based on gene neighbourhood analysis
The grouping of each of the ORFs into a specific cluster of amino acid sequence similarity allows the exploitation of the information on the chromosome environment where the HAA1 and CUP2 orthologues reside. Although the synteny information comprised in the YGOB database  already allows such analysis for most of the yeast species examined in this study, the genome sequences of Z. bailii species are still lacking in this database as well as the sequences of other species with the genomes currently available (S. paradoxus, S. arboricola, S. bayanus, Kluyveromyces marxianus var. Marxianus, K. wickerhamii, K. aestuarii and Ashbya aceri). For this reason, and since the combination of phylogenetic and comparative genomic analyses is a more reliable strategy to elucidate the orthologue/paralogue status of homologous genes, the analysis of the chromosome environment where all the HAA1 and CUP2 orthologue sequences reside was performed. Results confirm that these genes share many common neighbours (Fig. 7). Considering that putative Haa1/Cup2 transcription factors of protoploid Saccharomycetaceae species always show a protein size above 540 amino acids (average size of 622 amino acids), it is likely that the HAA1/CUP2 ancestral gene would encode a protein with size closer to the HAA1 orthologues. The gene neighbourhood analysis corroborated the indications obtained from classic phylogenetic methodologies that after the WGD event, a single gene lineage comprising the members of the Haa1/Cup2 family gave rise to two sub-lineages, each comprising the orthologues of S. cerevisiae HAA1 and CUP2 genes (Figs. 7 and 8). With the exception of the yeast species from Naumovozyma genus, all orthologues encoded in late-divergent post-WGD species share strong synteny (i.e. inside each sub-lineage). However, Vanderwaltozyma and Tetrapisispora species, belonging to early divergent taxonomic genera shortly differentiated after the WGD event, shows that their HAA1 and CUP2 orthologues share scarce synteny with the S. cerevisiae HAA1 and CUP2 genes and with other genes encoded in post-WGD species that evolved later, suggesting local genome-shuffling.
The single Haa1/Cup2 orthologue from Z. bailii is also involved in response and tolerance to copper stress
As previously demonstrated by Keller et al. , both S. cerevisiae BY4741 and derived haa1∆ mutant strains are more tolerant to CuSO4 than cup2∆ mutant strain that is very susceptible to this metal cation (Fig. 9a). On the other hand, S. cerevisiae BY4741 parental and derived cup2∆ mutant strains are remarkably more tolerant to acetic acid than haa1∆ mutant (Fig. 9c). Considering the proposed evolutionary history of Haa1 and Cup2 orthologues and their specific physiological functions in S. cerevisiae, the hypothesis of whether the sole ZbHaa1 could be involved in copper tolerance was tested. The heterologous expression of ZbHAA1 in S. cerevisiae cup2Δ mutant strain was found to rescue the susceptibility phenotype of the deletion mutant by increasing its tolerance to CuSO4 up to the level of the parental strain (Fig. 9a). Contrasting with these phenotypes, no growth was detected in minimal medium containing 0.15 mM CuSO4, either in S. cerevisiae cup2Δ transformed with the cloning vector or in the same background strain expressing ScHAA1 (Fig. 9a). The susceptibility of Z. bailii IST302 and derived deletion mutant Zbhaa1∆ to copper stress was also tested. Differently from the absence of alteration of the copper susceptibility phenotype by the deletion of ScHAA1 in S. cerevisiae, the deletion of ZbHAA1 in Z. bailii IST302 led to a marked increased susceptibility to copper (Fig. 9b). These results reinforce the concept that ZbHaa1 might have a dual function, being putatively involved in the regulation of acetic acid (as demonstrated above) and copper-responsive genes as found for its S. cerevisiae single orthologues Haa1 and Cup2, respectively.
In order to confirm the hypothesized role of ZbHaa1 in the response to copper, the transcript levels from S. cerevisiae metallothionein encoding gene CUP1, that is regulated by Cup2 under copper stress , were compared in S. cerevisiae cup2Δ mutant strain expressing ZbHAA1, in S. cerevisiae parental strain (positive control) and in the derived deletion mutant cup2Δ transformed with the cloning vector or expressing ScHAA1 (both used as negative controls). An increase in the mRNA level from CUP1 was observed in cells expressing ZbHAA1 after 1 and 2 h of exposure to 0.1 mM CuSO4 (Fig. 10). This increase was not detected in S. cerevisiae cup2Δ mutant strain either transformed with the cloning vector or expressing ScHAA1, demonstrating that S. cerevisiae CUP1 gene is regulated by ZbHaa1 under copper stress. Since no homologue of S. cerevisiae CUP1 could be identified in Z. bailii IST302 genome sequence, the homologue of S. cerevisiae metallothionein encoding gene CRS5, whose expression is dependent on CUP2 upon copper stress , was identified in the genome sequence of Z. bailii IST302 (ORF ZBIST_3713, here mentioned as ZbCRS5, Additional file 2). A remarkable increase in the mRNA levels from ZbCRS5 in Z. bailii IST302 parental strain was found during the early adaptive response to copper (1 h after sudden exposure to copper), being this activation of ZbCRS5 transcription significantly reduced in the Zbhaa1∆ deletion mutant (Fig. 11), indicating that the activation of ZbCRS5 is dependent on ZbHaa1 in Z. bailii cells under copper stress.
The physiological role of Z. bailii transcription factor ZbHaa1, homologous to S. cerevisiae Haa1, in the response and tolerance to acetic acid and copper was examined in this work by exploring functional and evolutionary analyses. It was demonstrated that ZbHaa1 is a functional homologue of ScHaa1, playing a role in Z. bailii tolerance to acetic acid and to other weak acid food preservatives. As previously reported for ScHaa1, the effect of ZbHaa1 is more evident for the more hydrophilic short-chain weak acid acetic acid . Moreover, similarly to the increased tolerance to acetic acid resulting from the overexpression of ScHAA1 in S. cerevisiae , the expression of an extra copy of ZbHAA1 in Z. bailii remarkably increases the tolerance of Z. bailii to all the weak acids tested. Furthermore, the involvement of ZbHaa1 in the transcriptional activation in response to acetic acid stress of Z. bailii genes homologous to S. cerevisiae Haa1-target genes reinforces the concept that ZbHaa1 is indeed a functional homologue of ScHaa1 and that the ZbHaa1-regulon is, at least in part, similar to the ScHaa1-regulon described before .
The identification of S. cerevisiae Haa1 homologues in Saccharomycetaceae yeast species showed that Z. bailii and all the other pre-WGD species do possess a sole Haa1 homologue that is also homologous to S. cerevisiae Cup2 , a transcription factor involved in yeast response and tolerance to high copper concentrations . The phylogenetic and gene neighbourhood analyses of the Haa1 and Cup2 homologues identified among 33 strains of 28 pre- and post-WGD Saccharomycetaceae yeast species allowed outlining their evolutionary history. A clear separation of the Haa1 and Cup2 homologues into three main groups resulted from the analysis: two groups containing either Haa1 or Cup2 orthologues encoded in the genomes of post-WGD yeast species, and a third containing Haa1/Cup2 orthologues from pre-WGD species. The only exception to the evolutionary pattern observed, in which post-WGD yeast species encode two members of the Haa1/Cup2 family in their genomes, is Tetrapisispora blattae that only possesses in the genome one member of this family. Since the genome sequencing was performed with high coverage (above 20X) and assembled into few scaffolds (10), it is likely that this yeast species may indeed encode a single member of this gene family. However, based on the size of the encoded protein (604 amino acids) and on its position in the phylogenetic tree, it was assigned as a Haa1 orthologue, although two neighbour gene families that also reside adjacent to the chromosomal region where the majority of Cup2 orthologues reside were found in its neighbourhood.
The evolutionary analysis performed reinforces the conclusion of previous studies suggesting that S. cerevisiae Haa1 and Cup2 are paralogues with origin in the WGD event [1, 19, 20]. After a gene duplication event, functional redundancy of the two duplicates is frequently avoided for their preservation in the genome and, eventually, fixation in the population gene pool [48–50]. Altogether, the indications obtained in this study led us to hypothesize that the sole ZbHAA1 gene could encode a bifunctional transcription factor controlling both the acetic acid and copper stress-response regulons that, upon the WGD event, subfunctionalized and partitioned the independent control of each regulon. In this study, experimental evidences are provided indicating that ZbHaa1 is indeed also involved in Z. bailii response and tolerance to copper stress. Moreover, the expression of ZbHAA1 in S. cerevisiae cup2∆ mutant leads to the transcriptional activation of the metallothionein encoding gene CUP1 that in S. cerevisiae is activated upon copper stress under the dependence of Cup2 transcription factor . Moreover, ZbHAA1 expression in Z. bailii significantly increases the transcription of ZbCRS5, a homologue of S. cerevisiae CRS5 gene that is also regulated by Cup2 transcription factor in copper-challenged cells . The fact that all genomes of the protoploid yeast species examined in this study encode a single Haa1/Cup2 orthologue suggests that the single Saccharomycetaceae yeast ancestral would have been a bifunctional transcription factor.
It is recognized that the duplication of transcription factors and/or of their gene targets is one of the most important evolutionary forces driving the evolution of transcriptional regulatory networks, leading to the expansion of existent networks or to the emergence of new ones, as well as allowing the acquisition of novel patterns of gene expression [50–52]. This study shows that the CUP2 orthologues experienced a series of deletion events throughout the evolution of the post-WGD species that led to a progressive decrease in the protein size. This reduction occurred essentially at the level of the transactivation domain while the DNA-binding domain remained highly conserved. Hence, the average protein size of 622 amino acids observed in the Haa1/Cup2 orthologues encoded in the genomes of the protoploid Saccharomycetaceae yeasts was reduced to a total of 341 amino acids in the Cup2 orthologue encoded in the genome of V. polysporus, a species that descended from an ancestral yeast that diverged immediately after the WGD event . Further deletions reduced the length of these proteins in late-diverging yeast species, with the S. cerevisiae Cup2 transcription factor showing a total size of 225 amino acids. These results support the hypothesis that after the WGD event this bifunctional ancestral gene divided its functions among the daughter duplicates through the acquisition of neutral degenerative mutations. Under this hypothesis, the observed indels in the amino acid sequences of the Cup2 orthologues would be explained by a specific subfunctionalization model, known as Duplication, Degeneration and Complementation . Other cases of subfunctionalization occurring in yeast and consistent with this model have been reported in the literature, for instance, for the paralogue pairs Gal1 and Gal3 , Sir2 and Hst1  and Bat1 and Bat2 . The completion of the subfunctionalization process that was in the origin of Haa1 and Cup2 is still unclear. Our results and those reported by Keller et al.  show that Haa1 is not involved in S. cerevisiae tolerance to copper. Although Cup2 was recently identified as a determinant of acetic acid tolerance in a quantitative trait loci analysis of pooled segregants that revealed the polygenic nature of high acetic acid tolerance in yeast, suggesting that in superior segregrants the CUP2 allele could take over the function of HAA1 , for S. cerevisiae S288c and under the experimental conditions used, we could not find evidences supporting the involvement of Cup2 in acetic acid tolerance. Our study points towards the evolution of both paralogues in a way that each one regulates a subset of the target genes all regulated by the single ancestral bifunctional transcription factor, given that ZbHaa1 activates Z. bailii genes homologous to both S. cerevisiae Haa1 and Cup2 target genes under acetic acid or copper-induced stresses. However, apparently, no cross-talk has been observed between both regulators in S. cerevisiae . S. cerevisiae Haa1 and Cup2 transcription factors recognize different motifs in promoter regions, respectively, 5’-(G/C)(A/C)GG(G/C)G-3’ and 5’-TC(T)4-6GCTG-3’  or 5’-HTHNNGCTGD-3’ . In this study it is demonstrated that ZbHaa1 can activate S. cerevisiae Haa1 and Cup2 target genes under acetic acid or copper stresses, respectively, which suggests that ZbHaa1 is apparently able to bind to the promoter regions of all those target genes. We have examined the promoters of the Z. bailii genes found in our study to be activated by ZbHaa1 under acetic acid or copper-induced stresses using the YEASTRACT database  and identified, in the promoter region of ZbHaa1 target genes ZbHRK1, ZbHSP30, ZbMSN4, ZbYRO2 and ZbCRS5, the presence of both S. cerevisiae Haa1 and Cup2 binding sites, but in ZbTPO3 and ZbYGP1 only the S. cerevisiae Haa1 recognition motif was found. Interestingly, the S. cerevisiae promoters of the Haa1 target genes YGP1, MSN4 and HSP30 and of the Cup2 target gene CRS5 also hold the binding sites for both Cup2 and Haa1 transcription factors. In order to understand how ZbHaa1 transcription factor regulatory network in Z. bailii relates with those regulated in S. cerevisiae by Haa1 and Cup2 transcription factors, it is crucial to determine the full ZbHaa1 regulon in Z. bailii and to elucidate the regulatory patterns of the target genes under copper or acetic acid-induced stresses. To get this information, it would be necessary at first to compare the transcriptomes of the parental Z. bailii strain and the mutant deleted for the gene encoding ZbHaa1 under those stresses and, subsequently, to fully identify the DNA motif(s) recognized by this transcription factor. Such information would provide insights for the understanding on how the DNA binding sequences have changed throughout evolution, and the mechanisms underlying the higher and lower specificity of the different orthologue proteins for the corresponding DNA binding motifs and subsequent transcriptional activation.
Based on functional and evolutionary analyses, this study demonstrates that the Z. bailii transcription factor homologous to S. cerevisiae Haa1 (ZbHaa1) is a bifunctional transcription factor, being required for response and tolerance to both acetic acid and copper stresses, and suggests the subfunctionalization of the ancestral Haa1/Cup2 orthologue after whole genome duplication, originating Haa1 and Cup2 paralogues. In S. cerevisiae, these genes were described as being individually involved in tolerance to acetic acid and copper stresses, respectively.
Acetic acid responsive element
Copper regulatory domain
DNA binding domain
Highest Posterior Density
Markov chain Monte Carlo
Message Passing Interface
Open reading frame
Polymerase chain reaction
Reverse transcription polymerase chain reaction
Saccharomyces genome database
Whole genome duplication
Yeast genome annotation pipeline
Yeast gene order browser
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This work was supported by Project contract PTDC/BBB-BEP/0385/2014 through “Fundação para a Ciência e a Tecnologia” (FCT)/MEC (PIDDAC) and PhD and Post-doctoral fellowships SFRH/BPD/73306/2010, SFRH/BPD/74618/2010, SFRH/BD/82226/2011 and SFRH/BD/80065/2011 awarded by FCT to MP, PJD, FCR and JFG, respectively. Funding received by iBB-Institute for Bioengineering and Biosciences from FCT (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged.
Availability of supporting data
All the supporting data of this article is included within the article and in its Additional files 1, 2, 3, 4 and 5. Nucleotide sequences of Z. bailii IST302 genes ZbHAA1, ZbHRK1, ZbHSP30, ZbTPO3, ZbMSN4, ZbYGP1, ZbYRO2 and ZbCRS5 have been deposited in GenBank® (http://www.ncbi.nlm.nih.gov) with the accession numbers KX656024, KX656025, KX656026, KX656027, KX656028, KX656029, KX656030 and KX656031, respectively.
MP performed the disruption of Z. bailii IST302 HAA1 homologue, cloning and expression of an extra copy of ZbHAA1 in Z. bailii IST302, susceptibility assays and comparison of mRNA relative levels in S. cerevisiae background strains. PJD performed the phylogenetic and gene neighbourhood analyses of the Haa1 homologues. FCR performed the comparison of mRNA relative levels in Z. bailii strains. JFG and LL cloned ZbHAA1 and ScHAA1 into pGREG506. MP, PJD and IS-C contributed to the writing of the manuscript. IS-C conceived and coordinated the study. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal
Margarida Palma, Paulo Jorge Dias, Filipa de Canaveira Roque, Laura Luzia, Joana Fernandes Guerreiro & Isabel Sá-Correia
Phylogenetic analysis of S. cerevisiae Haa1 and Cup2 transcription factors homologues encoded in the genomes of 33 strains of 28 yeast species belonging to the Saccharomycetaceae family. A) Radial phylogram showing the amino acid sequence similarity distances between these 51 full-size proteins. B) Circular cladogram showing the tree topology, with the name of the S. cerevisiae and Z. bailii members indicated. PhyML software suite was used in phylogenetic tree calculation. The gene and species annotation adopted in this study uses the four letters code described in Table 1. (PDF 1465 kb)
Gene neighbourhood analysis of the S. cerevisiae HAA1 and CUP2 orthologues from species of the Saccharomycetaceae family. Central boxes represent S. cerevisiae HAA1 and CUP2 orthologues. Adjacent boxes represent their gene neighbours. Homologous gene neighbours are highlighted using the same colour and identified with the same number. A white box represents genes with no homologous neighbours in the represented chromosome region. (PDF 2076 kb)
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Palma, M., Dias, P.J., Roque, F.d. et al. The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2.
BMC Genomics18, 75 (2017). https://doi.org/10.1186/s12864-016-3443-2