Eukaryotic transporters for hydroxyderivatives of benzoic acid

Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma membrane is currently unknown. In this study, we analyzed a family of genes coding for permeases belonging to the major facilitator superfamily (MFS) in the pathogenic yeast Candida parapsilosis. Our results revealed that these transporters are functionally equivalent to bacterial aromatic acid: H+ symporters (AAHS) such as GenK, MhbT and PcaK. We demonstrate that the genes HBT1 and HBT2 encoding putative transporters are highly upregulated in C. parapsilosis cells assimilating hydroxybenzoate substrates and the corresponding proteins reside in the plasma membrane. Phenotypic analyses of knockout mutants and hydroxybenzoate uptake assays provide compelling evidence that the permeases Hbt1 and Hbt2 transport the substrates that are metabolized via the gentisate (3-hydroxybenzoate, gentisate) and 3-oxoadipate pathway (4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate), respectively. Our data support the hypothesis that the carriers belong to the AAHS family of MFS transporters. Phylogenetic analyses revealed that the orthologs of Hbt permeases are widespread in the subphylum Pezizomycotina, but have a sparse distribution among Saccharomycotina lineages. Moreover, these analyses shed additional light on the evolution of biochemical pathways involved in the catabolic degradation of hydroxyaromatic compounds.


Results and Discussion
Identification of the genes for hydroxybenzoate transporters. Initially we attempted to search for candidate genes encoding the hydroxybenzoate transporters by blasting the C. parapsilosis genome using the amino acid sequences of bacterial transporters for aromatic acids classified into different superfamilies such as ABC, APC, MFS and OMPP (Supplementary Table S1). The searches with all but one bacterial query did not reveal any clear candidate for hydroxybenzoate carriers (E-values were above 10 −25 ). In case of the phthalate permease OphD from Burkholderia cepacia, the best identified hits were CPAR2_802720 and its three paralogs CPAR2_802710, CPAR2_802700 and CPAR2_802690 (E-values were 2 × 10 −35 to 2 × 10 −30 ). These genes are tandemly arranged on the chromosomal contig005807 and the analysis of deduced amino acid sequences predicted MFS_1 domain (PF07690) and twelve TMHs (except CPAR2_802690, which appears to be truncated at its N-terminus). Similarly to OphD, the C. parapsilosis proteins as well as the C. albicans CR_01220 W (an ortholog of CPAR2_802720) can be classified into the ACS family of MFS transporters 9,35 .
Next, we compared the expression of predicted plasma membrane transporter genes in cells assimilating 3-hydroxybenzoate, 4-hydroxybenzoate or glucose by RNA-seq analysis. Our results showed that the transporter gene with the highest level of expression in the control cells grown in synthetic medium containing glucose (SD) was CPAR2_212860 encoding an MFS protein with predicted sugar transporter domain (PF00083/Sugar_tr). Its ortholog in C. albicans is HGT7 (C2_01000 W) and codes for a glucose transporter 40,41 . We assumed that CPAR2_212860 may have similar function in C. parapsilosis. In contrast to SD medium, the most expressed genes in synthetic media containing a hydroxybenzoate are CPAR2_704330 (S3OH) and CPAR2_204840 (S4OH) that code for uncharacterized members of the MFS. These genes have no orthologs in C. albicans, which metabolizes hydroxybenzenes, but does not assimilate hydroxybenzoates, further supporting the idea that they are associated with the hydroxybenzoate metabolism. The expression of both genes was nearly undetectable on glucose, but they were highly upregulated in media containing a hydroxybenzoate (Supplementary Table S2). CPAR2_704330 was induced more than 1,402-fold on S3OH and CPAR2_204840 more than 1,247-fold on S4OH. Such strong induction was observed only on one hydroxybenzoate indicating that corresponding transporters could be specific for the substrate present in the cultivation medium.
Of the four OphD homologs identified by the BlastP searches, the highest level of expression has CPAR2_802720 on S4OH medium, where it exhibits 1,378-fold induction. In spite of such high induction, the overall transcript level of CPAR2_802720 was more than 14 times lower than observed for CPAR2_204840 and its paralogs (i.e. CPAR2_802710, CPAR2_802700 and CPAR2_802690) were weakly expressed on all three media (Supplementary Table S2).
BlastP searches using CPAR2_704330 and CPAR2_204840 as queries identified additional two uncharacterized MFS proteins, CPAR2_100470 and CPAR2_100460. These hits had E-values below 10 −80 , while all remaining hits had E-values above 10 −25 . The amino acid sequences of CPAR2_704330, CPAR2_204840, CPAR2_100470 and CPAR2_100460 display extensive sequence similarity ( Supplementary Fig. S1), possess a typical MFS_1 domain and twelve TMHs ( Supplementary Fig. S2). All four proteins belong to the Pfam clan CL0015 (Supplementary  Table S2). Previously, they were classified into the drug: H + antiporter 1 (DHA1) family (2.A.1.2) 37 . However, our phylogenetic analysis indicates that the sequences of all four C. parapsilosis proteins cluster with typical AAHS permeases ( Supplementary Fig. S3). Therefore, we propose that these proteins are eukaryotic members of the AAHS family (2.A.1.15). Their sequences exhibit less than 20% overall identity with the bacterial AAHS carriers such as BenK, GenK, PcaK and MhbT and lack a typical 'DGXD' motif present in the TMH 1. Nonetheless, a more detailed sequence comparison revealed several similarities with the bacterial transporters. The hydrophilic regions between the TMHs 2-3 and 8-9 contain conserved motifs 'VPXMX(R/A)YG(K/R)(R/K)' and 'G(Y/P) (M/L)SDX(L/W)(V/M)X(W/R)' , respectively ( Supplementary Fig. S4). These motifs resemble to the consensus 'GXXXD(R/K)XGR(R/K)' , which in bacterial MFS permeases has a role in the substrate transport 42 . Importantly, the aspartate residue in the motif present within the loop between TMHs 8 and 9, which is conserved in the entire AAHS family, occurs also in the C. parapsilosis transporters. In addition, charged residues R124, E144, R386 and R398 present in the sequence of Pseudomonas putida PcaK 43 were found also in the C. parapsilosis proteins (i.e. the arginines occur in the positions corresponding to R124 and R398; a glutamic acid is in E144 (except for CPAR2_704330, which has an aspartic acid in this position); and a lysine replaces R386). Moreover, the amino acid sequence alignment revealed several additional residues (i.e. G92, G165, P287, G310, G368 according to the PcaK numbering) that are shared by bacterial and C. parapsilosis proteins ( Supplementary Fig. S1). Importantly, some of the amino acid residues conserved between the bacterial AAHS and C. parapsilosis proteins were shown to be essential for activity of the AAHS transporters (Supplementary Table S3). Based on the sequence analysis and the experimental results shown below we named these genes as HBT1 (for hydroxybenzoate transporter 1; CPAR2_704330), HBT2 (CPAR2_204840), HBT3 (CPAR2_100470) and HBT4 (CPAR2_100460).
The HBT1 gene is located between the genes MNX2 (CPAR2_704320) and GDX1 (CPAR2_704340), within the metabolic gene cluster coding for the gentisate pathway enzymes 26 and the phylogenetic analysis showed that it co-evolved with other genes present in this cluster 39 further supporting its association with the gentisate pathway.
Phenotypic analysis of Δhbt mutants. The expression profiles of HBT genes indicated that corresponding protein products could be involved in the uptake of hydroxyaromatic compounds. To test this idea, we constructed a set of homozygous knockout strains each lacking a single HBT gene. In standardized phenotypic tests (Supplementary Table S5) we observed that the growth of Δhbt1/Δhbt1, Δhbt2/Δhbt2 and Δhbt3/Δhbt3 strains in complex (YPD) as well as synthetic media (SD 1% , SD 1% + FBS, YCB + BSA), the colony morphology, the formation of pseudohyphae, the sensitivity to oxidative stress (H 2 O 2 ), detergents (SDS), the inhibitors of cell wall biosynthesis (calcofluor white, congo red, caffeine), hygromycin B and antifungal drugs (caspofungin, fluconazole) remain unchanged when compared to the wild type strain. The phenotype of the Δhbt4/Δhbt4 mutant was similar, except its slower growth in synthetic media at 20 °C, formation of smaller colonies on SD 1% plates, resistance to caffeine and altered sensitivity to both antifungals (Supplementary Table S5, Supplementary Fig. S5).
Next, we tested the ability of mutant strains to grow in synthetic media containing a hydroxybenzoate as a sole carbon source (Fig. 2a,b). We observed impaired growth of the mutants Δhbt1/Δhbt1 and Δhbt2/Δhbt2 in media with substrates assimilated via the gentisate and 3-oxoadipate pathway, respectively. The phenotypes were more pronounced in media buffered to pH 7.5 ( Fig. 2b) possibly reflecting the fact that pKa values of all hydroxybenzoates are lower than 5. This may cause that in non-buffered media (at pH below 3.5) the hydroxyaromatic substrates are at least partially present in their undissociated form, which may enter the cells by a simple diffusion. In contrast to strains Δhbt1/Δhbt1 and Δhbt2/Δhbt2, the mutants Δhbt3/Δhbt3 and Δhbt4/Δhbt4 do not exhibit growth defect in these media. This may suggest that Hbt3 and Hbt4 are not involved in hydroxybenzoate uptake or their affinity to these compounds substantially differ from that of Hbt1 and Hbt2 (e.g. Hbt1 and Hbt2 may represent high-affinity and Hbt3 and Hbt4 low-affinity transporters). In the latter case, Hbt1 and/or Hbt2 would compensate the growth defects resulting from the absence of Hbt3 and Hbt4 proteins.
To verify that the phenotypes of Δhbt1/Δhbt1 and Δhbt2/Δhbt2 strains are caused by deletions of the transporter gene and to localize corresponding proteins within the C. parapsilosis cells, we transformed both mutants using the plasmids expressing the Hbt proteins tagged with the yEGFP3 at their C-termini. This experiment confirmed that pPK5-HBT1 and pPK5-HBT2 complement corresponding mutations. In addition, we observed that pPK5-HBT1 suppresses the growth defect also in the Δhbt2/Δhbt2 mutant suggesting that Hbt1 transports both 3-hydroxybenzoate and protocatechuate, although the native HBT1 gene is differentially regulated in cells assimilating these substrates ( Fig. 1). However, pPK5-HBT2 does not functionally complement the Δhbt1 mutation. We also found that neither Δhbt1/Δhbt1 nor Δhbt2/Δhbt2 cells transformed with the plasmid pPK5-HBT3 can grow in media containing 3-hydroxybenzoate (Δhbt1/Δhbt1) or protocatechuate (Δhbt2/Δhbt2) suggesting that Hbt3 does not participate in the uptake of these substrates (Fig. 3).
The examination of the transformants by fluorescence microscopy showed that the fusion proteins Hbt1-yEGFP3 and Hbt2-yEGFP3 are localized on the cell surface, presumably in the plasma membrane (Fig. 4).

Uptake of [ 14 C]-labeled hydroxybenzoates.
To investigate the transport of hydroxybenzoates into C. parapsilosis cells, we analyzed the uptake of [ 14 C]-labeled substrates (i.e. 3-hydroxybenzoate, 4-hydroxybenzoate and protocatechuate; Fig. 5). Our results showed that the wild type cells transport all three substrates, although the overall accumulated radioactivity in cells was substantially higher with [ 14 C]3-hydroxybenzoate than [ 14 C]4-hydroxybenzoate or [ 14 C]3,4-dihydroxybenzoate. These differences may at least partially result from different levels of HBT1-4 transcripts in the cells grown in S3OH, SHyd and S3,4diOH media ( Fig. 1, Supplementary  Table S2). Alternatively, corresponding transporters may exhibit different affinities to these substrates. The hydroxybenzoate uptake cannot be attributed to a simple diffusion as the assays were performed at pH 7.5, where about 99% of the substrate is present as a hydroxybenzoate anion that does not pass through the plasma membrane.
To examine if the transport of hydroxybenzoates is dependent on proton gradient, we treated the wild type cells with a protonophore (100 µM CCCP) prior addition of [ 14 C]3-hydroxybenzoate to the uptake assay. We found that CCCP almost completely inhibits the substrate uptake ( Table 1) thus confirming that the transport is driven by proton gradient across the plasma membrane.
In contrast to the wild type cells, the uptake of [ 14 C]3-hydroxybenzoate is almost completely abolished in the Δhbt1/Δhbt1 mutant (Fig. 5a). This indicates that Hbt1p is the major carrier for this substrate. The impaired growth of this mutant in S3OH and S2,5diOH (Fig. 2) further suggests that Hbt1p can transport both  Supplementary Table S4. 3-hydroxybenzoate as well as gentisate. The uptake of [ 14 C]3-hydroxybenzoate is decreased by 21, 26 and 29% also in Δhbt2/Δhbt2, Δhbt3/Δhbt3 and Δhbt4/Δhbt4 cells, respectively. The reason for reduced transport capacity of these mutants remains unclear. However, it seems unlikely that Hbt2, Hbt3 and Hbt4 contribute to the uptake of 3-hydroxybenzoate as the cells lacking Hbt1 apparently do not transport this substrate. To analyze the kinetics of 3-hydroxybenzoate transport, we examined the uptake of this substrate in eight concentrations ranging from 0.5 to 150 µM. Our results showed that 3-hydroxybenzoate is transported into the wild type cells with V max 0.63 ± 0.05 nmol/min/mg of dry weight and K m 21.58 ± 6.58 µM (Supplementary Fig. S6) indicating that, similarly to bacterial AAHS permeases (e.g. GenK, MhbT and PcaK) [12][13][14] , Hbt1 is a high-affinity transporter.
In most cases, the MFS permeases transport their substrates into cells along with H + ions 1, 44 . The hydroxyaromatic anion: H + symport systems were demonstrated in bacteria 12,45 as well as in basidiomycetes T. cutaneum (phenolate) 20 and F. palustris (vanillate) 22 . The amino acid sequence similarity of the Hbt proteins and bacterial AAHS transporters as well as the dependence of hydroxybenzoate uptake on the proton gradient led us to conclusion that C. parapsilosis Hbt proteins also function as proton symporters. We posit that Hbt1p and Hbt2p are involved in the uptake of hydroxybenzoates degraded via the gentisate and 3-oxoadipate pathway, respectively. The role of Hbt3p and Hbt4p remains elusive and will require further investigation.
Phylogeny of HBT genes and the evolution of hydroxybenzoate catabolism. Aromatic compounds derived from lignin in decaying plant tissues are catabolized via biochemical pathways operating in species from all three kingdoms of life (reviewed in ref. 46). In C. parapsilosis, the catabolism of hydroxybenzoates proceeds via the gentisate pathway and the hydroxyhydroquinone (HHQ) variant of the 3-oxoadipate pathway (Fig. 6). These pathways include activities of monooxygenases with broader substrate specificity (Mnx1, Mnx2, Mnx3) and dioxygenases (Hdx1, Gdx1) that open the aromatic ring of the catabolized compounds. Resulting products are then converted via several reactions to intermediates entering the central metabolism (e.g. tricarboxylic cycle). In contrast to C. parapsilosis, C. albicans lacks the gentisate pathway and possesses the catechol and HHQ variants of the 3-oxoadipate pathway 24 monooxygenase catalyzing the first step of the 3-oxoadipate pathway (i.e. Mnx1 in C. parapsilosis) and lacks homologs of the Hbt transporters, it can utilize hydroxybenzenes, but not hydroxybenzoates (Fig. 6). The difference between the two Candida species belonging to the CTG clade of Saccharomycotina could result from either a downgrade or an upgrade of corresponding metabolic pathways. In the first scenario, a common ancestor of the CTG clade species possessed the two branches of the 3-oxoadipate pathway as well as the gentisate pathway and it could catabolize both hydroxybenzoates and hydroxybenzenes. The gentisate pathway gene cluster and the genes coding for decarboxylating monooxygenase (Mnx1) and hydroxybenzoate transporters have been lost in the C. albicans lineage causing its inability to assimilate hydroxybenzoates. On the other hand, the C. parapsilosis lineage lost the genes for the catechol branch of the 3-oxoadipate pathway. Alternatively, an ancestor of both species possessed the catechol branch and a shorter version of the HHQ variant of the 3-oxoadipate pathway allowing the degradation of hydroxybenzenes (i.e. as it occurs in C. albicans) and the gentisate pathway. The genes encoding the enzymes of the latter pathway and the catechol variant of the 3-oxoadipate pathway have been lost in the lineages leading to C. albicans and C. parapsilosis, respectively. In the C. parapsilosis lineage, the duplications of HBT1 and MNX2 would generate HBT2 and MNX1, respectively, and led to an upgrade of the HHQ variant of the 3-oxoadipate pathway to allow the uptake and decarboxylation of hydroxybenzoates.
To examine these possibilities, we performed phylogenetic analysis of hydroxybenzoate transporters and their homologs (Fig. 7, see Methods). We found that the HBT1 orthologs are widespread in Pezizomycotina (e.g. Aspergillus and Penicillium species), but that their distribution among Saccharomycotina is very sparse. In the latter subphylum, the orthologs were identified in several species of the CTG clade (i.e. Candida maltosa, Debaryomyces hansenii, Debaryomyces fabryi, Lodderomyces elongisporus, Scheffersomyces stipitis, Spathaspora passalidarum) as well as in species classified outside of this phylogenetic branch (i.e. Brettanomyces bruxellensis, Kuraishia capsulata, Nadsonia fulvescens, Sugiyamaella lignohabitans). HBT2 orthologs show a more sparse distribution which is almost restricted to the CTG clade, with the sole exception of Wickerhamomyces anomalus. The distribution within the CTG clade only partially overlaps with that of Hbt1, being present in several common species (i.e. C. parapsilosis, D. hansenii, D. fabryi, L. elongisporus, S. passalidarum) and some additional species (C. orthopsilosis, M. guilliermondi). Importantly, our comparative and phylogenetic analyses suggest that Hbt1 and Hbt2 transporters show a high level of sequence divergence, and with the associations of these two clades receiving only weak phylogenetic support. Considering our obtained topology (Fig. 7a), the most parsimonious phylogenetic scenario suggests that Hbt1 and Hbt2 diverged within the Saccharomycotina but much before the origin of the CTG clade. Differential losses from a common ancestor would have resulted in the current sparse distribution. Finally, our analysis identified that the closer paralogs to HBT2 (i.e. HBT3 and HBT4) emerged from a specific duplication in the C. parapsilosis complex species (i.e. C. orthopsilosis and C. parapsilosis).
Vertical descent from a common ancestor of Saccharomycotina was proposed for evolution of the metabolic gene cluster encoding the gentisate and 3-oxoadipate pathway enzymes 38,39 . Phylogenetic distribution of the HBT paralogs correlates with the ability to grow on hydroxybenzoates. Hence, the duplication of HBT1 followed by functional specialization of HBT2 could extend the range of hydroxybenzoate substrates metabolized via the 3-oxoadipate pathway, supporting the upgrading scenario. However, our analysis indicates that this occurred a long time ago, before the divergence of the CTG clade from other Saccharomycotina, and that subsequent loss in some lineages resulted in the secondary loss of this ability.
As mentioned above, the upgrade of the 3-oxoadipate pathway requires a decarboxylation of a hydroxybenzoate substrate. In C. parapsilosis, this reaction is catalyzed by 4-hydroxybenzoate 1-hydroxylase (Mnx1), which has broad substrate specificity and utilizes 4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate 26,47 . This enzyme has a distant paralog Mnx2 (3-hydroxybenzoate 6-hydroxylase) catalyzing the first reaction of the gentisate pathway. The phylogenetic analysis of Mnx1 and Mnx2 indicates that their emergence from a common ancestor is also evolutionarily ancient and occurred before the divergence of the CTG clade from a Saccharomycotina ancestor. Based on the phylogenies of Hbt1-4 and Mnx1-2 proteins (Fig. 7) we assume that the ancestor possessed the gentisate pathway as well as the longer version of the 3-oxoadipate pathway, but had limited capacity for the hydroxybenzoate uptake as it possessed only the Hbt1 transporter. This limitation was overcome by the HBT1 gene duplication and subsequent specialization of the Hbt2 transporter. Many subsequent lineages have lost either of the pathways, given rise to their sparse distributions with little overlap that we see today. Thus, we propose a combined model, implying one ancient upgrade followed by multiple independent downgrades. Of note, HBT1 duplications seem to be common. However, the phylogenetic analysis indicates that these paralogs emerged independently of HBT2.

Conclusions
The yeast C. parapsilosis possesses MFS carriers Hbt1 and Hbt2 mediating the inducible proton driven transport system for hydroxybenzoates catabolized via the gentisate and 3-oxoadipate pathway, respectively. These transporters are functionally related to bacterial AAHS permeases and represent the first identified eukaryotic members of this family. Functional characterization of these carriers may contribute to exploration of yeast species in bioremediation of environments contaminated with toxic aromatic pollutants and utilization of compounds derived from lignin and decaying plant tissues.

Methods
Yeast cultivations. C. parapsilosis CLIB214 (identical to CBS604, the wild type strain) and its mutant derivatives were cultivated in synthetic and complex media listed in Table 2. The sensitivity to acidic and alkaline conditions was tested in YPD media with pH ranging from 4 to 8. pH of the respective media was adjusted using McIlvaine (citrate-phosphate) buffer. The temperature sensitivity was examined in YPD medium at 20, 30 and 37 °C for 48 hours. The growth kinetics was assessed in liquid YPD medium for 24 hours at 30 °C with hourly measurements at OD 600 . The viability was also monitored in the presence of various stressors applied in SCIENtIFIC REPORTS | 7: 8998 | DOI:10.1038/s41598-017-09408-6 serial twofold dilutions in six steps. The following stressors were used: calcofluor white (0.1 mg/ml), congo red (0.1 mg/ml), caffeine (0-50 mM), H 2 O 2 (7.5 mM), sodium dodecyl sulfate (SDS, 0.1% (w/v)) and hygromycin B (0.0156 mg/ml). The survival relative to the stressor-free control was monitored by measurements at OD 600 . The formation of pseudohyphae was analyzed in DMEM + FSB medium after 24 and 48 hours at 37 °C. In experiments aimed at the functional analysis of HBT genes, the cells were grown at 28 °C in synthetic media containing a hydroxyaromatic compound, glucose, galactose, glycerol or ethanol as a sole carbon source (Table 2). Hydroxyaromatic compounds were dissolved in dimethyl sulfoxide (DMSO) as 0.5 M stocks. Where indicated, pH of the medium was adjusted to 7.5 using 100 mM Tris-HCl prior addition of a carbon source. For solid media, agar was added to 2% (w/v).
Preparation of knockout strains. The mutants lacking an individual HBT gene were generated using a previously established method, adapted for C. parapsilosis 48,49 . Briefly, the disruption of a gene was achieved via auxotrophy complementation of the double auxotrophic strain named CPL2H1 (C. parapsilosis his − /leu − derived from CLIB214). Deletion constructions contained the upstream (UpFw primer 1 and UpRev primer 3;   48,49 , using the primer 2 and 5. Joining of the amplified products was achieved using polymerase chain reaction (PCR). Deletion cassettes were transformed into CPL2H1 strain and the transformants were plated onto selective media. Obtained heterozygous and homozygous mutant strains were verified by colony PCR using the primers specific for both the marker sequences and the outside of the integration sites at both the upstream and downstream homologous regions. Mutant strains were further tested for the gene expression using quantitative PCR (qPCR).
Gene expression analysis. Total cellular RNA was isolated from the culture of the wild type strain grown in synthetic media with appropriate carbon source by the phenol-chloroform extraction protocol 50 and the RNA preparations were treated with RNase-free DNase I (New England Biolabs) according to the manufacturer's instructions. The RNA-seq and real-time qPCR analyses were performed as described previously 39 . The sequencing reads have been deposited to Short Read Archive (PRJEB1707). The gene-specific primers used for qPCR assays are shown in Supplementary Table S6.
Plasmid constructs. The C. parapsilosis sequences coding for Hbt1, Hbt2 and Hbt3 proteins were amplified by PCR using the gene specific primers (Supplementary Table S6) and the template from the genomic DNA of the wild type strain. The PCR products containing the ORFs without the termination codon, plus 700 nucleotides upstream of the initiation codon, were inserted into the SalI site of the pPK5 vector 51 using the Gibson assembly cloning kit (New England Biolabs). The cloned genes containing native promoters are placed downstream of the GAL1 promoter in the resulting plasmid constructs.

Functional complementation of Δhbt mutants and fluorescence microscopy. Plasmid DNAs
were introduced into Δhbt cells by electroporation 52 and transformants were selected on media containing mycophenolic acid (SD + MPA). The utilization of hydroxybenzoates was tested in synthetic media containing glucose or a hydroxybenzoate as a sole carbon source. The cells transformed with the vector pPK5 were used as a negative control. For intracellular localization of Hbt proteins C-terminally tagged with yeast-enhanced green fluorescent protein 3 (yEGFP3), the transformants were cultivated overnight in SD + MPA medium at 28 °C, washed with water and the expression of fusion proteins was induced for 2-4.5 hours in SGal + MPA medium. Intracellular localization of Hbt1-yEGFP3 and Hbt2-yEGFP3 was investigated by fluorescence microscopy using BX50 microscope equipped with the appropriate filter set and digital camera DP70 (Olympus Optical).
Hydroxybenzoate uptake assays. C. parapsilosis cells were grown in synthetic media supplemented with hydroxyaromatic substrate (10 mM) as a sole carbon source (i.e. S3OH, S4OH and SHyd for the uptake of [ 14 C]3-hydroxybenzoate, [ 14 C]4-hydroxybenzoate and [ 14 C]3,4-dihydroxybenzoate, respectively) till late exponential phase and harvested by centrifugation (10 min, 3000 g at 4 °C). The pellet was washed once with ice-cold water and once with the assay buffer (50 mM Tris-HCl pH 7.5) and then resuspended in the same buffer at an OD 600 = 90. For each measurement, a 60 μl aliquot of cell suspension was used. The uptake assays were performed in the assay buffer at 28 Table 2. Cultivation media.
a [ 14 C]-labeled compound to a final concentration 50 μM. Aliquots (75 μl) were taken from the incubation mixture (364 μl) at timed intervals, immediately filtered through cellulose membranes (0.45-μm pore size; MF-Millipore) and washed twice with 4 ml ice-cold assay buffer. The amount of radioactivity accumulated in the cells was determined with a scintillation counter Tri-Carb 2900 TR (Perkin Elmer). The uptake activity was expressed as disintegrations per minute (dpm) per mg of cells (dry weight). The effect of a protonophore on the uptake of [ 14 C]3-hydroxybenzoate was tested in cells treated for two minutes with 100 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) prior addition of [ 14 C]-labeled substrate to the uptake assay. Apparent K m and V max values for the 3-hydroxybenzoate uptake were obtained essentially as described for bacterial GenK transporter 14 .
Phylogenetic analysis. Evolutionary histories of the genes considered were first visually inspected using the Maximum Likelihood phylogenies available in PhylomeDB 62 . Then individual phylogenies were reconstructed using these proteins and their closest blast hits in NCBI non-redundant database searched as of January 2017. The first 100 blast hits were used for MNX1 and MNX2 genes. For HBT1 and HBT2 this procedure did not render both genes within the first 100 hits of each other, but both lists contained common proteins. We thus performed the analysis of the combination of both lists. In brief, phylogenies were reconstructed as follows: protein sequences of the hits that passed a threshold of similarity (e-value < 10 −5 ) and coverage (>33% aligned over the query sequence), were aligned with MUSCLE 63 with default parameters, trimmed with trimAl v1.4 64 to eliminate alignment columns with more than 50% gaps. A Maximum Likelihood phylogenetic reconstruction was performed with PhyML v3 65 , using the LG model and approximating four rate categories and the fraction of invariable sites from the data. Then monophyletic clades containing the seed proteins and other Saccharomycotina species were selected. Duplicated sequences and pseudogenes were removed. Subtrees are shown in Fig. 7. Whole trees (in Newick format) are provided in Supplementary Files S1-S3.