FEMS Yeast Research, 15, 2015, fov006
doi: 10.1093/femsyr/fov006
Advance Access Publication Date: 5 March 2015
Research Article
RESEARCH ARTICLE
Metabolic gene clusters encoding the enzymes of two
branches of the 3-oxoadipate pathway in the
pathogenic yeast Candida albicans
Gabriela Gérecová1 , Martina Neboháčová1 , Igor Zeman1 , Leszek P. Pryszcz2,3 ,
Ľubomı́r Tomáška1 , Toni Gabaldón2,3,4 and Jozef Nosek1,∗
1
Departments of Biochemistry and Genetics, Faculty of Natural Sciences, Comenius University, Mlynska
dolina CH-1 and B-1, 842 15 Bratislava, Slovakia, 2 Bioinformatics and Genomics Programme, Centre for
Genomic Regulation (CRG), Doctor Aiguader 88, 08003 Barcelona, Spain, 3 Departament de Ciències
Experimentals I de la Salut, Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain and 4 Institució Catalana
de Recerca i Estudis Avançats (ICREA), Pg. Lluı́s Companys 23, 08010 Barcelona, Spain
∗ Corresponding author: Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1 842 15 Bratislava, Slovakia.
Tel: +421-2-60296-402; Fax: +421-2-60296-452; E-mail: [email protected]
One sentence summary: The genes for key enzymes involved in the degradation of phenolic compounds form two clusters in the Candida albicans
genome.
Editor: Carol Munro
ABSTRACT
The pathogenic yeast Candida albicans utilizes hydroxyderivatives of benzene via the catechol and hydroxyhydroquinone
branches of the 3-oxoadipate pathway. The genetic basis and evolutionary origin of this catabolic pathway in yeasts are
unknown. In this study, we identified C. albicans genes encoding the enzymes involved in the degradation of
hydroxybenzenes. We found that the genes coding for core components of the 3-oxoadipate pathway are arranged into two
metabolic gene clusters. Our results demonstrate that C. albicans cells cultivated in media containing hydroxybenzene
substrates highly induce the transcription of these genes as well as the corresponding enzymatic activities. We also found
that C. albicans cells assimilating hydroxybenzenes cope with the oxidative stress by upregulation of cellular antioxidant
systems such as alternative oxidase and catalase. Moreover, we investigated the evolution of the enzymes encoded by these
clusters and found that most of them share a particularly sparse phylogenetic distribution among Saccharomycotina,
which is likely to have been caused by extensive gene loss. We exploited this fact to find co-evolving proteins that are
suitable candidates for the missing enzymes of the pathway.
Keywords: evolution of biochemical pathways; catabolism of phenols; gene clustering; gene expression; comparative
genomics
INTRODUCTION
Phenol and its derivatives are highly toxic environmental
pollutants. Although these compounds exhibit strong antimicrobial activity, there are bacterial and fungal species able to
utilize them as growth substrates employing several metabolic
pathways (Harwood and Parales 1996; Fuchs, Boll and Heider
2011). Under aerobic conditions, phenols are degraded via the 3oxoadipate (β-ketoadipate) pathway, where a monooxygenasecatalyzed hydroxylation of the substrate is followed by the cleavage of the aromatic ring by a dioxygenase. Resulting products
Received: 3 October 2014; Accepted: 2 February 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2015, Vol. 15, No. 3
are then metabolized via the tricarboxylic acid (TCA) cycle.
Pioneering studies on the yeasts Candida tropicalis and Trichosporon cutaneum revealed that both species completely
degrade phenol via catechol branch of the 3-oxoadipate pathway (Neujahr, Lindsjo and Varga 1974; Gaal and Neujahr 1979;
Mortberg and Neujahr 1985; Krug and Straube 1986). In this
branch, ortho cleavage of catechol produces cis,cis-muconate,
which is cyclized to muconolactone. Its isomerization into
3-oxoadipate enol-lactone is followed by formation of
3-oxoadipate. On the other hand, hydroquinone and resorcinol are metabolized via hydroxyhydroquinone (HHQ; 1,2,
4-trihydroxybenzene, hydroxyquinol) branch of the 3oxoadipate pathway. These dihydroxybenzenes are hydroxylated to HHQ, which is cleaved to 2-maleylacetate followed
by its reduction to 3-oxoadipate. Both branches merge at
3-oxoadipate, which is further transformed to succinate and
acetyl-CoA (Fig. 1). Previous studies revealed that yeast species
from the monophyletic ‘CTG clade’ of hemiascomycetes differ
in the range of assimilated phenolic compounds (Middelhoven
et al. 1992; Middelhoven 1993; Holesova et al. 2011), thus providing an excellent platform for investigation of corresponding
biochemical pathways by means of comparative genomics. For
example, C. albicans utilizes phenol, catechol, hydroquinone
and resorcinol as sole carbon sources, indicating that both
branches of the 3-oxoadipate pathway operate in this species.
In contrast, C. parapsilosis apparently lacks the catechol branch
as it does not assimilate phenol and catechol. Yet, C. parapsilosis
grows on a range of hydroxybenzoates and, in addition to the
3-oxoadipate pathway, it employs the gentisate pathway for
catabolism of 3-hydroxybenzoate and 2,5-dihydroxybenzoate.
The genetic basis of these physiological differences remains
mostly uncharacterized.
Recently, we have identified the genes coding for key
enzymes of the 3-oxoadipate and gentisate pathways in
C. parapsilosis. These genes are highly induced in media
containing corresponding hydroxyaromatic substrate (i.e.
3-hydroxybenzoate and 4-hydroxybenzoate induce the gentisate and 3-oxoadipate pathway genes, respectively) and these
two pathways are controlled independently at the transcriptional level (Holesova et al. 2011). In addition, we found that the
gentisate pathway genes are arranged into a cluster coding for
3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase,
fumarylpyruvate hydrolase, a major facilitator superfamily
transporter, a binuclear Zn(2)Cys(6) transcription factor and
a protein exhibiting homology to glutathione-dependent
formaldehyde-activating enzyme.
As both C. albicans and C. parapsilosis belong to the same
phylogenetic clade, their distinct abilities to assimilate phenolic
compounds provide a platform for exploration of the genetic
basis of underlying metabolism. With this goal, we analyzed
the genetic control of hydroxybenzene degradation in C. albicans. First, we identified homologs of the genes coding for key
components of the 3-oxoadipate pathway and found that they
are organized into two metabolic gene clusters corresponding
to the catechol and HHQ branches of this pathway. Next, we
analyzed the expression of corresponding genes in C. albicans
cells assimilating hydroxybenzene substrates using real-time
quantitative polymerase chain reaction (qPCR) and assessed
the enzymatic activities by NAD(P)H oxidation and oxygen
consumption assays. In addition, we investigated the activity
of cellular antioxidant systems such as alternative oxidase
and catalase whose induction accompanies the catabolism of
hydroxybenzene compounds. Finally, we studied the evolution
of the enzymes in the pathway and searched for co-evolving
genes that may code for the enzymatic activities whose genetic
basis remains to be identified.
MATERIALS AND METHODS
Yeast strains and cultivation
Yeasts C. albicans CBS562NT , C. dubliniensis CBS7987T , C. glabrata
CBS138T , C. orthopsilosis MCO457T , C. parapsilosis CBS604T and
C. tropicalis CBS94T were used in assimilation tests of hydroxyaromatic compounds. Yeasts were cultivated at 28◦ C in
minimal synthetic media containing 0.67% (w/v) of yeast nitrogen base w/o amino acids (Difco), 2% (w/v) glucose (SD) or 10 mM
hydroxybenzene derivative [i.e. phenol (SPhe), catechol (SCat),
hydroquinone (SHyd), resorcinol (SRes), 2-hydroxybenzoate
(S2OH), 3-hydroxybenzoate (S3OH), 4-hydroxybenzoate (S4OH),
2,3-dihydroxybenzoate
(S2,3diOH),
2,4-dihydroxybenzoate
(β-resorcylate,
S2,4diOH),
2,5-dihydroxybenzoate
(gentisate, S2,5diOH) and 3,4-dihydroxybenzoate (protocatechuate,
S3,4diOH)]. Hydroxyaromatic compounds were dissolved in
dimethylsulfoxide (DMSO) as 500 mM stocks. Solid media
contained 2% agar.
Bioinformatic analyses
We used the enzyme amino acid sequences of the HHQ branch
from C. parapsilosis [i.e. Mnx1/CPAR2 102790 (4-hydroxybenzoate
1-hydroxylase), Mnx3/CPAR2 205970 (hydroquinone hydroxylase), Hdx1/CPAR2 406440 (hydroxyquinol 1,2-dioxygenase),
Osc1/CPAR2 406450
(3-oxoadipate
CoA-transferase)
and
Oct1/CPAR2 212970 (3-oxoadipyl-CoA thiolase)] and the catechol branch from Aspergillus nidulans [i.e. AN7418 (phenol
2-monoxygenase), AN4532 (catechol 1,2-dioxygenase), AN3895
(muconate isomerase), AN4061 (muconolactone isomerase) and
AN4531 (3-oxoadipate enol-lactonase)] as queries in database
searches. Since the gene coding for maleylacetate reductase and
most of the catechol branch enzymes have not been identified
in Candida species, we searched the C. albicans proteome also
with the sequences of maleylacetate reductases from Burkholderia (tftE/Uniprot acc.no.: Q45072), Pseudomonas (clcE/O30847,
tcbF/P27101), Ralstonia (macA/Q9RBG1, tfdF/P94135) and
Rhodococcus (macA/O84992), and the catechol branch enzymes
from Pseudomonas putida [i.e. catA/Q52041 (catechol 1,2dioxygenase), catB/P08310 (cis,cis-muconate cycloisomerase),
catC/P00948 (muconolactone isomerase), pcaD/M1LCW2
(3-oxoadipate enol-lactonase), pcaI/Q01103, pcaJ/P0A102
(subunits of 3-oxoadipate CoA-transferase) and pcaF/Q51956
(3-oxoadipyl-CoA thiolase)]. The gene sequences of C. albicans
and their systematic names were obtained from Candida
Genome Database (http://www.candidagenome.org/; Inglis
et al. 2012). Amino-acid sequences were aligned using MAFFT
v7.017 algorithm (Katoh et al. 2002) and conserved domains in
predicted proteins were identified using InterProScan (Jones
et al. 2014). The synteny and orthologous genes were examined
using the Candida Gene Order Browser (http://cgob3.ucd.ie/;
Fitzpatrick et al. 2010; Maguire et al. 2013). Intracellular localization of identified proteins was predicted by MitoProt
(http://ihg.gsf.de/ihg/mitoprot.html; Claros and Vincens 1996)
and PSORT II (http://psort.hgc.jp/; Nakai and Horton 1999).
Phylogenetic profiling and analysis
Phylogenetic profiles of C. albicans proteins were generated by
performing BlastP searches (Altschul et al. 1990) against 69
Saccharomycetes species, 10 Aspergillus and 6 Fusarium proteomes available in MetaPhOrs database as of June 2014 (Pryszcz,
Gérecová et al.
hydroquinone
resorcinol
OH
phenol
OH
OH
OH
OH
hydroquinone hydroxylase
orf19.604 / PHH1
and/or
orf19.3711 / PHH2
phenol 2-monooxygenase
orf19.604 / PHH1
and/or
orf19.3711 / PHH2
NAD(P)H
+ O2
NAD(P)H
+ O2
NAD(P)H
+ O2
1,2,4-trihydroxybenzene (HHQ)
catechol
OH
OH
OH
OH
OH
catechol 1,2-dioxygenase
orf19.4567 / HQD2
O2
hydroxyquinol 1,2-dioxygenase
orf19.2282 / HQD1
cis,cis-muconate
O2
2-maleylacetate
C OOH
C OOH
O
cis,cis-muconate cycloisomerase
orf19.4571 / MCI1
C OOH
C OOH
muconolactone
maleylacetate reductase
NAD(P)H
O
HOOC
O
muconolactone
isomerase
orf19.4570 / MLI1
3-oxoadipate
3-oxoadipate enol-lactone
O
HOOC
O
O
C O OH
C OOH
3-oxoadipate enol-lactonase
orf19.4569 / OEL1
3-oxoadipate CoA-transferase
orf19.2281 / OSC1
succinyl-CoA
succinate
3-oxoadipyl-CoA
S-CoA
HOOC
O
3-oxoadipyl-CoA thiolase
orf19.2046 / POT1-2
O
CoA
succinyl-CoA
acetyl-CoA
Figure 1. Catabolic degradation of hydroxybenzenes in C. albicans via two branches of the 3-oxoadipate pathway.
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FEMS Yeast Research, 2015, Vol. 15, No. 3
Huerta-Cepas and Gabaldón 2011). Presence of a Blast hit below a
stringent threshold of (e-value < 10−25 ) was considered as presence of a homolog in that genome, otherwise the protein was
considered to be absent. More relaxed thresholds (10−10 , 10−5 )
often resulted in detection of a relatively large fraction of false
positives in the phylogenetic profiles. Note that the stringent
threshold is sufficient to detect the Pezizomycotina orthologs
for all known components of the pathway, so we do not expect a high rate of false negatives within the Saccharomycotina.
Proteins with a similar phylogenetic profile as those identified
in the cluster were detected by computing the Hamming distance to the profile of Hqd1. Hamming distance between two
profiles measures the minimum number of changes in presence/absence calls required to change one phylogenetic profile
into the other (Gabaldón 2008). Phylogenetic histories of the relevant genes were inspected using the maximum-likelihood phylogenies available in PhylomeDB (Huerta-Cepas et al. 2014).
Gene expression analysis
Candida albicans cells were grown aerobically in 250 mL of liquid
synthetic media till the late exponential phase, and total RNA
was isolated essentially as described by Cross and Tinkelenberg
(1991). RNA samples were treated with RNase-free DNase I (New
England Biolabs) and cDNA was synthesized using Maxima H
Minus First Strand cDNA Synthesis Kit with oligo(dT) primers
(Thermo Scientific). The qPCR analyses were performed on
cDNA templates using Luminaris Color HiGreen High ROX
qPCR Master Mix (Thermo Scientific) in a StepOne cycler
(Applied Biosystems). Gene-specific primers are listed in Table
S1 (Supporting Information). All reactions were carried out
in technical duplicates and normalized to the mean levels of
transcripts of two housekeeping genes ACT1 and EFB1. Relative
mRNA levels for all genes were determined in three independent experiments and calculated by relative standard curve
method. The differences between the gene expression in cells
grown in media containing a hydroxybenzene substrate and SD
medium were evaluated by Student’s t-test (∗ P < 0.05; ∗∗ P < 0.01;
∗∗∗
P < 0.001).
NAD(P)H oxidation assay
Candida albicans cultures were grown till the late exponential
phase in synthetic media. Cells were then harvested by centrifugation (10 min, 3000 g at 4◦ C), washed with water and resuspended in 5 volumes 50 mM Tris-HCl, 5 mM EDTA (pH 8.0). The
suspension was homogenized in a FastPrep 24 cell disrupter using 1.2 g mL−1 of lysing matrix C (MP Biomedicals) three times
for 20 s at a speed setting of 6.5 m s−1 and the lysates were centrifuged (10 min, 1500 g at 4◦ C). Substrate-dependent enzymatic
activities present in the supernatant were measured essentially
as described by Eppink et al. (1997). Briefly, 67–333 μg of proteins
were added to 50 mM potassium phosphate buffer (pH 7.6) containing 0.2 mM NADPH, 10 mM FAD and 1 mM of appropriate
substrate. Oxidation of NADPH at 340 nm and 25◦ C was continuously monitored by using a Varian Cary 50 Bio UV/Visible spectrophotometer. The amount of oxidized NADPH was calculated
from the decrease in the absorbance at defined time intervals.
One unit of enzyme activity is defined as the amount of enzyme
catalyzing the oxidation of 1 nmol of NADPH per minute at 25◦ C.
Oxygen consumption assay
Candida albicans grown in synthetic media was harvested by centrifugation (10 min, 2500 g at 4◦ C), washed twice with water,
then with 40 mM potassium phosphate buffer (pH 7.4) and re-
suspended in the same buffer. Oxygen consumption was measured at 30◦ C in a thermostatically controlled chamber equipped
with a Clark electrode. Oxygen depletion by endogenous respiration and after addition of 10 mM hydroxybenzene substrates was recorded and analyzed by using an oxygen meter
782 with data analysis module (Strathkelvin Instruments). The
respiratory chain enzymes were inhibited using antimycin A
(8 μg mL–1 ), KCN (10 mM), rotenone (4 μM) or salicylhydroxamate
(SHAM; 2 mM).
Catalase activity assay
Total catalase activity was determined by the method of
Roggenkamp, Sahm and Wagner (1974). The cell extracts were
prepared as described above, except that the homogenization
was performed in ice-cold 50 mM potassium phosphate buffer
(pH 7.0). For each assay, 2.5–10 μg of proteins were mixed with
1.5–4.5 mM hydrogen peroxide and the rate of its decomposition
was measured spectrophotometrically at 240 nm (ε = 43.6 cm−1
mol−1 dm3 ) using a Varian Cary 50 Bio UV/Visible spectrophotometer. One unit of catalase activity is defined as the amount of
enzyme catalyzing the degradation of 1 μmol of H2 O2 per minute
at 25◦ C. Alternatively, the method of Woodbury, Spencer and
Stahmann (1971) was used. Cell extracts (5–20 μg of proteins)
were electrophoretically separated in non-denaturing 5% polyacrylamide gels. The gels were then soaked in 10 mM hydrogen
peroxide for 10 min, rinsed in water and stained in a solution of
freshly prepared 2% (w/v) potassium ferricyanide and 2% (w/v)
ferric chloride.
RESULTS
Candida albicans assimilates hydroxybenzenes, but not
hydroxybenzoates
We examined the range of hydroxyaromatic compounds utilized by C. albicans in synthetic media containing 10 mM hydroxyderivatives of benzene or benzoic acid as sole carbon
sources (Fig. 2, Fig. S1, Supporting Information). For comparison,
we included additional five Candida species in the assimilation
test (Table S2, Supporting Information). With the exception of
C. glabrata, which does not belong to the CTG-clade but is
rather closer to Saccharomyces (Marcet-Houben and Gabaldón
2009), all examined yeasts metabolize hydroxyaromatic compounds. Candida albicans, its sibling species C. dubliniensis and
C. tropicalis utilize phenol and all three dihydroxybenzenes, but
do not grow in media with hydroxybenzoates as sole carbon
sources. In contrast, C. parapsilosis and C. orthopsilosis assimilate
hydroquinone and resorcinol as well as a range of hydroxybenzoates. None of the species grows in media containing salicylate
or 2,3-dihydroxybenzoate. Assimilated hydroxyaromatic compounds are metabolized via the catechol (e.g. phenol, catechol)
and HHQ (e.g. hydroquinone, resorcinol, 4-hydroxybenzoate)
branches of the 3-oxoadipate pathway or by the gentisate
pathway (e.g. 3-hydroxybenzoate, gentisate). The range of assimilated compounds indicates that the HHQ branch of the
3-oxoadipate pathway operates in most examined species, while
the catechol branch is present in C. albicans, C. dubliniensis and
C. tropicalis, and the gentisate pathway is limited to C. parapsilosis. Although C. albicans as well as C. dubliniensis and C. tropicalis
exhibit functional 3-oxoadipate pathways, the inability to grow
in media with hydroxybenzoates (e.g. 4-hydroxybenzoate) suggests that these species are unable to transport, decarboxylate
and/or cleave the aromatic ring of these derivatives.
Gérecová et al.
5
hydroxylase (Mnx3) from C. parapsilosis (Eppink et al. 2000) and
phenol 2-monooxygenase from C. tropicalis (Krug and Straube
1986). We have also noticed that the catechol-dependent activity
predominates in cells assimilating phenol as well as dihydroxybenzenes (Fig. 3C–F). This goes in line with the observation of
Tsai, Tsai and Li (2005) that catechol 1,2-dioxygenase from C. albicans exhibits higher activity than phenol 2-monooxygenase at
lower concentrations of phenol (5–15 mM).
Metabolic gene clusters for the 3-oxoadipate pathway
enzymes
Figure 2. Assimilation of hydroxyderivatives of benzene and benzoic acid
by C. albicans CBS562. Cell suspensions were spotted in serial 10-fold dilutions onto plates with synthetic media containing 2% glucose (SD) or 10
mM phenol (SPhe), catechol (SCat), resorcinol (SRes), hydroquinone (SHyd),
2-hydroxybenzoate (S2OH), 3-hydroxybenzoate (S3OH), 4-hydroxybenzoate
(S4OH), 2,3-dihydroxybenzoate (S2,3diOH), 2,4-dihydroxybenzoate (S2,4diOH),
2,5-dihydroxybenzoate (S2,5diOH) and 3,4-dihydroxybenzoate (S3,4diOH) as a
sole carbon source and cultivated at 28◦ C for 1 day (SD) or 3–7 days (remaining
media). The growth curves of cultures in media with hydroxybenzene substrates
are shown in Fig. S1 (Supporting Information). For assimilation profiles of additional Candida species, see Table S2 (Supporting Information).
Next, we examined the enzymatic activities involved in
the metabolism of hydroxybenzenes in C. albicans. As several enzymes of the 3-oxoadipate pathway are dependent on
NAD(P)H (i.e. phenol 2-monooxygenase, hydroquinone hydroxylase and maleylacetate reductase) or molecular oxygen (i.e.
phenol 2-monooxygenase, hydroquinone hydroxylase, catechol
1,2-dioxygenase and hydroxyquinol 1,2-dioxygenase) (Fig. 1), we
used NAD(P)H oxidation and oxygen consumption assays for
monitoring both branches of this pathway in C. albicans (Fig. 3).
NAD(P)H oxidation assays were performed with protein extracts
prepared from cells grown in synthetic media containing glucose (SD) or a hydroxybenzene as a sole carbon source. The
extracts prepared from cells cultivated in SD medium exhibited relatively low NADPH oxidation after addition of hydroxyaromatic substrates. However, we detected 35-, 98- and 82fold increase of NADPH oxidation in the extracts from cells
grown in media with 10 mM phenol (SPhe), resorcinol (SRes)
and hydroquinone (SHyd), respectively (Fig. 3A). Similarly, we
observed that substrate-dependent oxygen consumption is significantly higher [from 1.9- (resorcinol) to 11.3-fold (catechol)]
in cells cultivated in media containing a hydroxybenzene substrate than in SD medium (Fig. 3B–F). These results demonstrate that the enzymes catalyzing reactions of the 3-oxoadipate
pathway are induced in cells assimilating hydroxybenzenes. We
found that the cells grown in SPhe medium display increased
oxygen consumption with substrates from both branches of
the 3-oxoadipate pathway (Fig. 3C). This suggests that both
pathways are co-activated and/or the corresponding enzymes
have broader substrate specificity, similar to hydroquinone
To identify C. albicans genes coding for the enzymes involved
in the metabolism of phenolic compounds, we used the
amino-acid sequences of fungal as well as bacterial enzymes
as queries in BlastP searches (Kasberg et al. 1995; Jiménez et al.
2002; Seibert et al. 2004; Holesova et al. 2011; Martins et al. 2015).
In addition, we analyzed the syntenic context of identified
genes using the Candida Gene Order Browser (Fitzpatrick
et al. 2010; Maguire et al. 2013). Our analysis revealed that
C. albicans genome contains homologs of C. parapsilosis and A.
nidulans genes coding for the enzymes of both branches of the
3-oxoadipate pathway (Fig. S2, Supporting Information; Table 1).
We identified orthologs of C. parapsilosis MNX3 (orf19.604/PHH1),
HDX1 (orf19.2282/HQD1), OSC1 (orf19.2281/OSC1) and OCT1
(orf19.2046/POT1-2). The searches with A. nidulans queries
revealed orthologs of AN7418 (orf19.3711/PHH2), AN4532
(orf19.4567/HQD2), AN3895 (orf19.4571/MCI1) and AN4061
(orf19.4570/MLI1). Moreover, we found that AN4531 shows high
similarity to orf19.4569/OEL1, a protein with alpha/beta hydrolase fold indicative of 3-oxoadipate enol-lactonase. However,
the BlastP searches did not reveal any candidate gene coding for
maleylacetate reductase. Moreover, we found no homologs of
C. parapsilosis MNX1 coding for 4-hydroxybenzoate 1hydroxylase nor the genes for the gentisate pathway enzymes,
which is consistent with the assimilation profile described above
(Fig. 2) and explains the inability of C. albicans cells to utilize
hydroxyderivatives of benzoic acid such as 3-hydroxybenzoate,
4-hydroxybenzoate, β-resorcylate, gentisate or protocatechuate.
The HQD1 locus has conserved synteny with the HDX1
region in C. parapsilosis genome, which encodes hydroxyquinol
1,2-dioxygenase (Holesova et al. 2011). It has been demonstrated previously that HQD2 (a paralog of HQD1) codes for
catechol 1,2-dioxygenase (Tsai and Li 2007). This indicates
that the identified genes code for the enzymes belonging to
parallel branches of the 3-oxoadipate pathway (Fig. 1). We
noticed that the genes, with the exception of PHH1, PHH2 and
POT1-2, are arranged into two clusters orf19.2280–orf19.2284
and orf19.4567–orf19.4571, which are conserved in a number
of the ‘CTG clade’ species (Fig. 4). The first cluster codes for
a protein with flavin reductase domain (orf19.2284), putative
dehydroquinate dehydratase (orf19.2283/DQD1), hydroxyquinol
1,2-dioxygenase (HQD1), 3-oxoadipate CoA-transferase (OSC1)
and a zinc cluster transcription factor (orf19.2280/ZCF10).
The counterpart of this cluster in the genomes of
C. parapsilosis, C. metapsilosis, C. orthopsilosis and Lodderomyces
elongisporus lacks a homolog of DQD1 gene. The second cluster
encodes catechol 1,2-dioxygenase (HQD2), a zinc cluster transcription factor (orf19.4568/ZCF25), 3-oxoadipate enol-lactonase
(OEL1) and the orthologs of A. nidulans muconolactone isomerase (MLI1) and muconate isomerase (MCI1), which contains
CoA-transferase family III domain. The occurrence of the gene
clusters in the ‘CTG clade’ species genomes correlates with their
ability to assimilate hydroxybenzenes (Table S2, Supporting
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FEMS Yeast Research, 2015, Vol. 15, No. 3
Figure 3. Enzymatic activities involved in the 3-oxoadipate pathway analyzed by NAD(P)H oxidation (A) and oxygen consumption (B–F) assays. Candida albicans cells
were grown in synthetic media containing 2% glucose (SD) or 10 mM hydroxybenzene (SPhe, SCat, SRes, SHyd) as a sole carbon source and enzymatic activities were
determined as described in the section ‘Materials and Methods’. (A) Monooxygenase activities inferred from NADPH oxidation in protein extracts were measured after
addition of 0.2% DMSO or 1 mM hydroxybenzene (i.e. phenol, resorcinol and hydroquinone). (B–F) Oxygen consumption was monitored using Clark oxygen electrode
after addition of 2% DMSO, 2% glucose or 10 mM hydroxybenzene (i.e. phenol, catechol, resorcinol and hydroquinone). The assays were performed in three independent
experiments with two parallel measurements in each case (error bars, mean ± SEM). The differences between the samples and the control (DMSO) were evaluated by
Student’s t-test (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001).
Information). The yeast species utilizing hydroquinone and
resorcinol possess the first cluster, while the second cluster
occurs only in species assimilating phenol and catechol. This
goes in line with the idea that the first cluster codes for core
enzymes of the HHQ branch and the second one for the catechol
branch enzymes of the 3-oxoadipate pathway.
The results of NADPH oxidation and oxygen consumption assays (Fig. 3) indicated that the 3-oxoadipate pathway enzymes
are activated in cells utilizing hydroxybenzene substrates, but
not in cells grown in glucose medium. Therefore, we investigated the expression of the identified genes in cells cultivated
in synthetic media containing glucose, phenol, catechol, hydroquinone or resorcinol as a sole carbon source by qPCR analysis.
Our results revealed that, besides DQD1 and orf19.2284, the expression of the examined genes in the SD medium is low, but upregulated in cells utilizing hydroxybenzene compounds (Fig. 5;
Table S3, Supporting Information). For example, the expression
of PHH1 and PHH2 in SPhe versus SD medium is increased
>150- and >3800-fold, respectively. The expression of genes
present in the clusters is induced on substrates of both branches
of the 3-oxoadipate pathway (i.e. phenol/catechol and resorcinol/hydroquinone). Relative expressions of individual genes
vary on different hydroxyaromatic substrates, although the gene
expression profiles in cultures from SRes and SHyd media are
similar.
Catabolism of hydroxybenzenes is accompanied by
upregulation of cellular antioxidant systems
The products of the 3-oxoadipate pathway, succinate and acetylCoA, enter into central metabolic pathways such as the TCA
cycle. Hence, yeast cells metabolizing phenolic compounds require functional mitochondria. In C. albicans, mitochondria possess intricate respiratory pathways comprising a conventional
respiratory chain with three phosphorylation-coupling sites,
a parallel respiratory chain and inducible cyanide-resistant
b
a
orf19.604
orf19.3711
orf19.2284
orf19.2283
orf19.2282
orf19.2281
orf19.2280
orf19.4567
orf19.4568
orf19.4569
orf19.4570
orf19.4571
orf19.2046
PHH1
PHH2
DQD1
HQD1
OSC1
ZCF10
HQD2
ZCF25
OEL1
MLI1
MCI1
POT1-2
Hamming distances were calculated with respect to the phylogenetic profile of Hqd1.
Alignments of amino-acid sequences and identified protein domains are shown in Fig. S2 (Supporting Information).
c
Based on predictions using MitoProt (http://ihg.gsf.de/ihg/mitoprot.html; Claros and Vincens 1996) and PSORT II (http://psort.hgc.jp/; Nakai and Horton 1999).
d
Best hit (e-value <10−25 ) in BlastP searches.
Cytoplasm
Cytoplasm
Mitochondria
Cytoplasm
Cytoplasm
Mitochondria
Nucleus
Cytoplasm
Nucleus
Mitochondria
Cytoplasm
Cytoplasm
Mitochondria
92.8
88.7
93.9
98.6
93.9
95.4
83.7
91.7
83.7
90.9
87.0
91.8
94.1
AN7418d
AN7418
AN7922
qutE AN1135
AN0764
AN5669
qutA AN1134
AN4532
None
AN4531d
AN4061
AN3895
AN4009
ORF
Gene
8
8
15
14
0
3
19
0
20
9
18
4
50
MNX3 CPAR2 205970
MNX3d CPAR2 205970
CPAR2 406430
none
HDX1 CPAR2 406440
OSC1 CPAR2 406450
CPAR2 406460
HDX1d CPAR2 406440
None
None
None
None
OCT1 CPAR2 212970
74.3
71.9
76.0
94.5
80.4
85.7
50.1
76.2
53.0
59.0
52.7
69.8
84.0
64.3
61.0
56.6
66.7
81.5
41.1
76.8
Phenol 2-monooxygenase and/or hydroquinone hydroxylase
Phenol 2-monooxygenase and/or hydroquinone hydroxylase
Unknown, has FMN-binding/flavin reductase domain
3-dehydroquinate dehydratase
Hydroxyquinol 1,2-dioxygenase
3-oxoadipate CoA-transferase
Zn(II)2 Cys6 transcription factor
Catechol 1,2-dioxygenase
Zn(II)2 Cys6 transcription factor
3-oxoadipate enol-lactonase
Muconolactone isomerase
cis,cis-muconate cycloisomerase (CoA-transferase family III)
3-oxoadipyl-CoA thiolase
localizationc
C. parapsilosis
C. tropicalis
C. dubliniensis
ortholog
ortholog
distancea
Identity of amino acid sequences [%]b
A. nidulans
C. parapsilosis
Hamming
C. albicans
Table 1. The list of C. albicans genes potentially involved in the metabolism of hydroxybenzenes.
Inferred enzymatic activity
Intracellular
Gérecová et al.
7
alternative oxidase (Helmerhorst et al. 2002; Ruy, Vercesi and
Kowaltowski 2006). We examined the effect of the respiratory
pathways inhibitors on the oxygen consumption in cells assimilating a hydroxybenzene compound (resorcinol). We inhibited
the respiratory complex I and cytochrome bc1 complex using
rotenone and antimycin A, respectively. We also used KCN at 10
mM concentration, which inactivates both the conventional and
parallel respiratory pathways, and SHAM blocking the activity of
alternative oxidase. Our results showed that cells metabolizing
resorcinol consume oxygen mostly by respiration (Fig. 6). The
inhibitors decreased oxygen consumption to 82.5% (rotenone),
57.4% (antimycin A), 88.4% (SHAM) and 18.3% (KCN) of the untreated control. In cells where all three respiratory pathways
were inhibited by using a combination of SHAM and KCN, the
oxygen consumption was reduced to 14.1%, which can be assigned to activities of hydroquinone hydroxylase and hydroxyquinol 1,2-dioxygenase. However, the oxygen consumption by
the 3-oxoadipate pathway enzymes is probably higher, as Eppink
et al. (2000) have shown that hydroquinone hydroxylase from C.
parapsilosis is partially inhibited by monovalent anions including
CN− , which compete with the aromatic substrate, and 20 mM
KCN decreases the enzyme activity to about 50%.
The sensitivity to SHAM indicates that the alternative oxidase is active in cells metabolizing hydroxybenzenes. Candida
albicans has two paralogs orf19.4774/AOX1 and orf19.4773/AOX2
encoding this enzyme (Huh and Kang 2001). We observed that
their expression is induced 86- and 259-fold, respectively, in cells
assimilating resorcinol (Fig. 7) as well as other hydroxybenzenes
(Table S3, Supporting Information). This goes in line with the role
of alternative oxidase to reduce the formation of reactive oxygen species (ROS; Helmerhorst et al. 2002; Li et al. 2011). Next,
we analyzed the activity of catalase, another cellular antioxidant system. In the C. albicans genome, this enzyme is encoded
by a single gene orf19.6229/CAT1 (Wysong et al. 1998). Our results showed that the catalase activity is induced more than 10fold in cells metabolizing a hydroxybenzene substrate (Fig. 8).
Upregulation of alternative oxidase and catalase activities apparently reduces the oxidative stress in cells degrading hydroxyaromatic compounds. This cellular response may have a clinical importance as it has been reported that the endogenous ROS
production mediates the activity of antifungals such as azoles
and the induction of alternative oxidase decreases susceptibility of C. albicans to these agents (Kobayashi et al. 2002; Yan et al.
2009). Moreover, the induction of catalase activity on hydroxybenzene substrates may have a protective effect against hydrogen peroxide-induced stress, similarly as reported for yeasts
treated with salicylates (Yiannakopoulou and Tiligada 2009).
Evolutionary history of hydroxybenzene assimilation
pathways
Proteins involved in the same metabolic pathway tend to coevolve (Gabaldón and Huynen 2004). Therefore, we searched
for proteins with a similar phylogenetic distribution as Hqd1
by computing the Hamming distances among their phylogenetic profiles (Gabaldón 2008). Table S4 (Supporting Information) lists 180 proteins with a Hamming distance of 20 or lower
with respect to the 85 species phylogenetic profile of Hqd1.
About one-third of these genes are differentially expressed
in biofilms and almost 20% are regulated by CCAAT-binding
complex-dependent transcription factor Hap43. The top seven
proteins in the list (Hqd1, Hqd2, Osc1, Mci1, Phh1, Phh2 and Oel1)
are clearly involved in the 3-oxoadipate pathway, and Hqd1 has
an identical profile as Hqd2, suggesting both branches of the
8
FEMS Yeast Research, 2015, Vol. 15, No. 3
Cluster I
C. albicans / C. dubliniensis / C. tropicalis / D. hansenii / Sch. stipitis / Sp. passalidarum
orf19.2283
orf19.2284
orf19.2282
orf19.2281
orf19.2280
DQD1
OSC1
ZCF10
HQD1
C. parapsilosis / C. metapsilosis / C. orthopsilosis / L. elongisporus
CPAR2_406430
CPAR2_406440
CPAR2_406450
HDX1
CPAR2_406460
OSC1
Cluster II
C. albicans / C. dubliniensis / C. tropicalis / D. hansenii / Sch. stipitis / Sp. passalidarum
orf19.4567
orf19.4568
orf19.4570
orf19.4569
HQD2
ZCF25
OEL1 MLI1
orf19.4571
MCI1
1 kbp
dioxygenase
flavin reductase
CoA transferase
3-dehydroquinate dehydratase
muconate isomerase (CoA transferase family III)
Zinc cluster transcription factor
muconolactone isomerase
3-oxoadipate enol-lactonase
Figure 4. Metabolic gene clusters coding for the 3-oxoadipate pathway enzymes identified in the genome of C. albicans. Cluster I and II encode core components of the
HHQ and catechol branch, respectively. A comparative analysis revealed that both clusters are conserved in the genomes of related yeast species from the ‘CTG’ clade.
Figure 5. Relative mRNA expression of C. albicans genes potentially involved in the metabolism of hydroxybenzenes. Candida albicans cells were grown in SD, SPhe,
SCat, SRes and SHyd media. Quantification of mRNA levels by qPCR was performed as described in the section ‘Materials and Methods’. The assays were performed
in three independent experiments with two parallel replicates in each case (error bars, mean ± SEM) and the results were evaluated by Student’s t-test (∗ P < 0.05;
∗∗
P < 0.01; ∗∗∗ P < 0.001). Induction of the gene expression (fold change) is shown in Table S3 (Supporting Information).
pathway have co-evolved to some extent. The list also contains
additional proteins encoded by the two metabolic gene clusters
(Dqd1, orf19.2284, Mli1, Zcf10 and Zcf25), secreted lipases (Lip1–
Lip10), proteins with predicted monooxygenase activity (Ifk2,
Fmo1 and Fmo2), members of cytochrome P450 (CYP) superfamily (Alk6, Alk8, orf19.7512, Alk2, orf19.5728 and orf19.1411) and
mitochondrial respiratory chain enzymes (e.g. subunits of NADH
dehydrogenase, alternative oxidase) pointing to their possible
association with the catabolism of hydroxybenzenes. In particular, the deduced protein product of orf19.2284 encoded by
the HHQ branch cluster has a flavin reductase domain found in
NAD(P)H-flavin oxidoreductases and predicted N-terminal mitochondrial import signal (Table 1). Moreover, it is expressed
on hydroxybenzene-containing media (Fig. 5), which makes it
a strong candidate for maleylacetate reductase. Next, the three
putative dimethylaniline monooxygenases Ifk2, Fmo1 and Fmo2
have FAD/NAD(P)-binding and monooxygenase domains like
Phh1, Phh2 and C. parapsilosis Mnx3. Thus, they are good candidates to act on some substrates of the pathway. Alternatively, dimethylaniline or its derivative could be degraded via
the catechol branch, as it has been reported for aniline in some
Candida species (Mucha et al. 2010; Wang et al. 2011). Similarly,
Gérecová et al.
Figure 6. The effect of respiratory chain inhibitors on the oxygen consumption
of cells metabolizing a hydroxybenzene substrate. Candida albicans cells were
grown in synthetic medium containing resorcinol as a carbon source (SRes),
washed and resuspended in potassium phosphate buffer as described in the section ‘Materials and Methods’. Oxygen consumption was monitored using Clark
oxygen electrode after addition of 10 mM resorcinol (the control) and respiratory
chain inhibitors rotenone (4 μM), antimycin A (8 μg mL–1 ), SHAM (2 mM), KCN (10
mM) or a combination of SHAM and KCN. The inhibitory effects are expressed
as a percentage of the untreated control. The assays were performed in three
independent experiments with two parallel measurements in each case (error
bars, mean ± SEM).
Figure 7. Relative mRNA expression of C. albicans genes AOX1 and AOX2 coding
for alternative oxidase. Candida albicans cells were grown in SD and SRes media.
Quantification of mRNA levels by qPCR was performed as described in the section ‘Materials and Methods’. The assays were performed in three independent
experiments with two parallel replicates in each case (error bars, mean ± SEM)
and the results were evaluated by Student’s t-test (∗∗ P < 0.01). Induction of the
gene expression (fold change) is shown in Table S3 (Supporting Information).
Figure 8. Catalase activity is induced in C. albicans assimilating hydroxybenzenes. Candida albicans cells were grown in SD, SHyd and SCat media and the
catalase activity has been determined in cell extracts spectrophotometrically
(A) or by staining of native gels (B) as described in the section ‘Materials and
Methods’. The assays were performed in three independent experiments with
two parallel measurements in each case (error bars, mean ± SEM). The significance of differences between the samples (SHyd, SCat) and the control (SD) was
evaluated by Student’s t-test (∗ ∗ P < 0.01; ∗ ∗ ∗ P < 0.001).
9
the members of the CYP superfamily may participate in the catechol branch, in parallel with phenol 2-monooxygenase, as it
has been demonstrated that inducible microsomal CYP hydroxylates phenol to catechol in C. tropicalis (Stiborova et al. 2003).
To assess whether the patchy phylogenetic distribution of
the proteins encoded in the gene clusters was the result of
putative horizontal transfer events or, alternatively, differential
gene loss, we inspected maximum-likelihood phylogenetic
reconstructions available in PhylomeDB C. albicans phylome
205 (Huerta-Cepas et al. 2014). Here we present the Hqd1/Hqd2
phylogenetic tree as an example (Fig. 9). HQD1 and HQD2 are
distant homologs, which diverged after an ancient duplication that predated the divergence of the two main Dikarya
groups: basidiomycetes and ascomycetes. Their distribution
is broad among Pezizomycotina but very restricted in Saccharomycotina, being present in the ‘CTG clade’ species (Fig. 10).
The long branches separating the Candida Hqd1/Hqd2 from
their Pezizomycotina counterparts, as well as their separated position as a clearly differentiated clade rather than
one deeply embedded within the Pezizomycotina, favor the
hypothesis of a vertical inheritance from a common Pezizomycotina/Saccharomycotina ancestor rather than a transfer from
Pezizomycotina to Saccharomycotina. This implies that the two
pathways have been retained from the ancestral Saccharomycotina in the lineages leading to few extant Saccharomycotina
species, while being lost in several Saccharomycotina lineages such as the common ancestor of Saccharomycetaceae
(Saccharomyces/Klyuveromyces/Lachancea). Molecular phylogenies
of the remaining components of the clusters as seen in PhylomeDB phylome 205 show similar histories, with only minor
differences in terms of their presence among extant ascomycete
species. In general, all enzymes of the pathway are widespread
in several subphyla from both Ascomycota and Basidiomycota.
Although some of the genes are missing in some few species of
these fungal phyla, the Saccharomycotina subphylum presents
a clear patchy distribution shared by most members of the
pathway.
DISCUSSION
Clustering of functionally associated genes into operons is a
typical feature of bacterial genomes. Such arrangement allows
coordinated synthesis of several gene products from a single
polycistronic transcript. Although operons are rarely present in
eukaryotes (Pettitt et al. 2014) and eukaryotic genes appear scattered in a genome, the frequent occurrence of gene clusters indicates that the eukaryotic gene order is not random (Hurst, Pal
and Lercher 2004). A number of gene clusters encoding the enzymes of intermediary and secondary metabolism have been
identified in fungal genomes (Keller and Hohn 1997; Wong and
Wolfe 2005; Hall and Dietrich 2007; Jeffries and Van Vleet 2009;
Fitzpatrick et al. 2010; Holesova et al. 2011; Greene et al. 2014;
Wisecaver, Slot and Rokas 2014). Such biochemical pathways
are usually dispensable, but they may provide a growth advantage during specific circumstances. Typical metabolic gene clusters comprise the genes for functionally linked enzymes involved in a biochemical pathway as well as for transcription
factors mediating coordinated and/or high-level gene expression. At the same time, the gene clustering facilitates transmission of the whole biochemical pathway by horizontal gene
transfer and contributes to extensive metabolic diversity of fungal species and their adaptation to the changing environment
(Hall and Dietrich 2007; Slot and Rokas 2011; Wisecaver, Slot
and Rokas 2014). A total of 38 metabolic gene clusters were
10
FEMS Yeast Research, 2015, Vol. 15, No. 3
Figure 9. Molecular phylogeny of Hqd1/Hqd2 family as reported in PhylomeDB phylome 205, which is based on genomes of 60 fully sequenced fungi.
The whole tree structure is shown schematically, with lineages colored according to their taxonomic classification: Basidiomycetes in blue, Pezizomycotina
in red and Saccharomycotina in yellow. The ancient gene duplication mentioned in the text is indicated with a red circle. Saccharomycotina-containing
subtrees are shown in more detail, to show the name of the species. The whole tree can be interactively browsed at PhylomeDB (URL for the tree:
http://phylomedb.org/?q=search tree&seqid=Phy0002J38&phid=205).
identified in the genome of C. albicans using the Kyoto Encyclopedia of Genes and Genomes analysis (Fitzpatrick et al.
2010). As most of them represent core metabolic pathways,
the overall number of gene clusters may be underestimated.
In this study, we described additional two gene clusters coding for enzymes involved in catabolic degradation of hydroxybenzenes, which are conserved among species classified into
the ‘CTG clade’ of hemiascomycetes (Fig. 4). These clusters
encode core components of the HHQ and catechol branch
of the 3-oxoadipate pathway including dioxygenases (Hqd1
and Hqd2), cis,cis-muconate cycloisomerase (Mci1), muconolactone isomerase (Mli1), 3-oxoadipate enol-lactonase (Oel1), 3oxoadipate CoA-transferase (Osc1), a protein with flavin reductase domain (orf19.2284) representing a candidate for maleylacetate reductase as well as binuclear Zn(II)2 Cys6 transcription
factors (Znf10 and Znf25), which are potentially involved in the
transcriptional control of the 3-oxoadipate pathway. Biochemical assays (Fig. 3) as well as qPCR analysis (Fig. 5) showed that
the 3-oxoadipate pathway enzymes and corresponding genes
exhibit low expression in cells cultivated in glucose medium, but
they are induced in cells assimilating hydroxybenzenes.
We noted that most identified genes displayed a patchy
distribution, being present only in some species among
Saccharomycotina (Fig. 10), and having homologs within Pezizomycotina, particularly in Aspergillus and Fusarium species.
We exploited this patchy distribution to detect other proteins
that co-evolved with known components of the 3-oxoadipate
pathway and which constitute clear candidates for the missing
enzymes. Our analysis highlights the power of comparative
genomics to generate specific and testable predictions of
the function of uncharacterized genes (Gabaldón and Huynen 2004). Of note, the arrangement of the two C. albicans
clusters is not conserved in Pezizomycotina species. For example, in A. nidulans, the genes for the 3-oxoadipate pathway enzymes are scattered in the genome, except for the
gene pair AN4531 and AN4532 representing the counterparts of OEL1 and HQD2, respectively, and orthologs of ZCF10
(qutA/AN1134) and DQD1 (qutE/AN1135), which belong to quinic
acid utilization cluster (Hawkins et al. 1988). On the other
hand, Fusarium oxysporum contains several gene clusters presumably involved in degradation of hydroxyaromatic compounds (e.g. FOZG 10520-FOZG 10530; FOZG 12162-FOZG 12164;
FOZG 13935-FOZG 13937; FOZG 15223-FOZG 15228).
In summary, our analysis of the two metabolic gene clusters identified in the genome of C. albicans provides an insight
into evolution of the 3-oxoadipate pathway in yeasts. Further
Gérecová et al.
11
Figure 10. Phylogenetic profile showing the presence and absence of the 3-oxoadipate pathway genes across several sequenced Saccharomycotina species. The phylogenetic tree is based on the analysis of a concatenated alignment of one-to-one orthologs present in all the included species, as determined by MetaPhOrs approach
(Pryszcz, Huerta-Cepas and Gabaldón 2011). Species translating CUG codon as serine (‘CTG clade’) are denoted with color background: haploid species in light blue
and diploid species in dark blue. Presence and absence profiles of the 3-oxoadipate pathway genes are denoted with the following colors: black (no ortholog), violet
(exactly one ortholog) and red (two orthologs).
studies may delineate the evolutionary trajectories leading to
clustering of functionally associated genes and reveal the means
for integration of hydroxybenzene catabolism with the central
metabolic pathways in eukaryotic cells.
SUPPLEMENTARY DATA
Council under the European Union’s Seventh Framework Programme (FP/2007-2013) / ERC (Grant Agreement n. ERC-2012StG-310325). LPP is funded through La Caixa-CRG International
Fellowship Program.
Conflict of interest. None declared.
Supplementary data is available at FEMSYR online.
REFERENCES
ACKNOWLEDGEMENTS
We thank Ladislav Kovac and Jordan Kolarov for continuous support and members of our laboratories for discussions.
FUNDING
This work was supported by the Slovak grant agencies VEGA
(1/0405/11 and 1/0311/12) and APVV (0123-10 and 0035-11) to JN
and LT. TG group was supported in part by a grant from the
Spanish ministry of Economy and Competitiveness (BIO201237161), a Grant from the Qatar National Research Fund grant
(NPRP 5-298-3-086), and a grant from the European Research
Altschul SF, Gish W, Miller W, et al. Basic local alignment search
tool. J Mol Biol 1990;215:403–10.
Claros MG, Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences.
Eur J Biochem 1996;241:779–86.
Cross FR, Tinkelenberg AH. A potential positive feedback loop
controlling CLN1 and CLN2 gene expression at the start of
the yeast cell cycle. Cell 1991;65:875–83.
Eppink MH, Boeren SA, Vervoort J, et al. Purification and
properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent
monooxygenase from Candida parapsilosis CBS604. J Bacteriol
1997;179:6680–7.
12
FEMS Yeast Research, 2015, Vol. 15, No. 3
Eppink MH, Cammaart E, Van Wassenaar D, et al. Purification and properties of hydroquinone hydroxylase, a FADdependent monooxygenase involved in the catabolism of 4hydroxybenzoate in Candida parapsilosis CBS604. Eur J Biochem
2000;267:6832–40.
Fitzpatrick DA, O’Gaora P, Byrne KP, et al. Analysis of gene evolution and metabolic pathways using the Candida Gene Order
Browser. BMC Genomics 2010;11:290.
Fuchs G, Boll M, Heider J. Microbial degradation of aromatic
compounds—from one strategy to four. Nat Rev Microbiol
2011;9:803–16.
Gaal A, Neujahr HY. Metabolism of phenol and resorcinol in
Trichosporon cutaneum. J Bacteriol 1979;137:13–21.
Gabaldón T. Comparative genomics-based prediction of protein
function. Methods Mol Biol 2008;439:387–401.
Gabaldón T, Huynen MA. Prediction of protein function and
pathways in the genome era. Cell Mol Life Sci 2004;61:930–44.
Greene GH, McGary KL, Rokas A, et al. Ecology drives the distribution of specialized tyrosine metabolism modules in fungi.
Genome Biol Evol 2014;6:121–32.
Hall C, Dietrich FS. The reacquisition of biotin prototrophy in Saccharomyces cerevisiae involved horizontal gene transfer, gene
duplication and gene clustering. Genetics 2007;177:2293–307.
Harwood CS, Parales RE. The beta-ketoadipate pathway and the
biology of self-identity. Annu Rev Microbiol 1996;50:553–90.
Hawkins AR, Lamb HK, Smith M, et al. Molecular organisation of
the quinic acid utilization (QUT) gene cluster in Aspergillus
nidulans. Mol Gen Genet 1988;214:224–31.
Helmerhorst EJ, Murphy MP, Troxler RF, et al. Characterization of
the mitochondrial respiratory pathways in Candida albicans.
Biochim Biophys Acta 2002;1556:73–80.
Holesova Z, Jakubkova M, Zavadiakova I, et al. Gentisate and
3-oxoadipate pathways in the yeast Candida parapsilosis:
identification and functional analysis of the genes coding for
3-hydroxybenzoate 6-hydroxylase and 4-hydroxybenzoate
1-hydroxylase. Microbiology 2011;157:2152–63.
Huerta-Cepas J, Capella-Gutiérrez S, Pryszcz LP, et al. PhylomeDB
v4: zooming into the plurality of evolutionary histories of a
genome. Nucleic Acids Res 2014;42:D897–902.
Huh WK, Kang SO. Characterization of the gene family encoding alternative oxidase from Candida albicans. Biochem J
2001;356:595–604.
Hurst LD, Pal C, Lercher MJ. The evolutionary dynamics of eukaryotic gene order. Nat Rev Genet 2004;5:299–310.
Inglis DO, Arnaud MB, Binkley J, et al. The Candida genome
database incorporates multiple Candida species: multispecies search and analysis tools with curated gene and protein information for Candida albicans and Candida glabrata.
Nucleic Acids Res 2012;40:D667–74.
Jeffries TW, Van Vleet JR. Pichia stipitis genomics, transcriptomics, and gene clusters. FEMS Yeast Res 2009;9:793–807.
Jiménez JI, Miñambres B, Garcı́a JL, et al. Genomic analysis of
the aromatic catabolic pathways from Pseudomonas putida
KT2440. Environ Microbiol 2002;4:824–41.
Jones P, Binns D, Chang HY, et al. InterProScan 5: genome-scale
protein function classification. Bioinformatics 2014;30:1236–
40.
Kasberg T, Daubaras DL, Chakrabarty AM, et al. Evidence that
operons tcb, tfd, and clc encode maleylacetate reductase, the
fourth enzyme of the modified ortho pathway. J Bacteriol
1995;177:3885–9.
Katoh K, Misawa K, Kuma K, et al. MAFFT: a novel method for
rapid multiple sequence alignment based on fast Fourier
transform. Nucleic Acids Res 2002;30:3059–66.
Keller NP, Hohn TM. Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol 1997;21:17–29.
Kobayashi D, Kondo K, Uehara N, et al. Endogenous reactive oxygen species is an important mediator of miconazole antifungal effect. Antimicrob Agents Ch 2002;46:3113–7.
Krug M, Straube G. Degradation of phenolic compounds by the
yeast Candida tropicalis HP 15. II. Some properties of the
first two enzymes of the degradation pathway. J Basic Microb
1986;26:271–81.
Li D, Chen H, Florentino A, et al. Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is
associated with increased reactive oxidants and cell death.
Eukaryot Cell 2011;10:672–82.
Maguire SL, ÓhÉigeartaigh SS, Byrne KP, et al. Comparative
genome analysis and gene finding in Candida species using
CGOB. Mol Biol Evol 2013;30:1281–91.
Marcet-Houben M, Gabaldón T. The tree versus the forest: the
fungal tree of life and the topological diversity within the
yeast phylome. PLoS One 2009;4:e4357.
Martins TM, Hartmann DO, Planchon S, et al. The old 3oxoadipate pathway revisited: new insights in the
catabolism of aromatics in the saprophytic fungus Aspergillus nidulans. Fungal Genet Biol 2015;74:32–44.
Middelhoven WJ. Catabolism of benzene compounds by ascomycetous and basidiomycetous yeasts and yeastlike fungi.
A literature review and an experimental approach. Anton
Leeuw 1993;63:125–44.
Middelhoven WJ, Coenen A, Kraakman B, et al. Degradation of
some phenols and hydroxybenzoates by the imperfect ascomycetous yeasts Candida parapsilosis and Arxula adeninivorans: evidence for an operative gentisate pathway. Anton
Leeuw 1992;62:181–7.
Mortberg M, Neujahr HY. Uptake of phenol by Trichosporon cutaneum. J Bacteriol 1985;161:615–9.
Mucha K, Kwapisz E, Kucharska U, et al. Mechanism of aniline
degradation by yeast strain Candida methanosorbosa BP-6. Pol
J Microbiol 2010;59:311–5.
Nakai K, Horton P. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization.
Trends Biochem Sci 1999;24:34–6.
Neujahr HY, Lindsjo S, Varga JM. Oxidation of phenols by cells
and cell-free enzymes from Candida tropicalis. Anton Leeuw
1974;40:209–16.
Pettitt J, Philippe L, Sarkar D, et al. Operons are a conserved
feature of nematode genomes. Genetics 2014;197:
1201–11.
Pryszcz LP, Huerta-Cepas J, Gabaldón T. MetaPhOrs: orthology
and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score. Nucleic
Acids Res 2011;39:e32.
Roggenkamp R, Sahm H, Wagner F. Microbial assimilation of
methanol induction and function of catalase in Candida boidinii. FEBS Lett 1974;41:283–6.
Ruy F, Vercesi AE, Kowaltowski AJ. Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation. J Bioenerg Biomembr
2006;38:129–35.
Seibert V, Thiel M, Hinner IS, et al. Characterization of a gene
cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4fluorobenzoate. Microbiology 2004;150:463–72.
Slot JC, Rokas A. Horizontal transfer of a large and highly toxic
secondary metabolic gene cluster between fungi. Curr Biol
2011;21:134–9.
Gérecová et al.
Stiborova M, Sucha V, Miksanova M, et al. Hydroxylation of
phenol to catechol by Candida tropicalis: involvement of cytochrome P450. Gen Physiol Biophys 2003;22:167–79.
Tsai SC, Li YK. Purification and characterization of a catechol 1,2dioxygenase from a phenol degrading Candida albicans TL3.
Arch Microbiol 2007;187:199–206.
Tsai SC, Tsai LD, Li YK. An isolated Candida albicans TL3 capable
of degrading phenol at large concentration. Biosci Biotech Bioch
2005;69:2358–67.
Wang D, Zheng G, Wang S, et al. Biodegradation of aniline by Candida tropicalis AN1 isolated from aerobic granular sludge. J Environ Sci 2011;23:2063–8.
Wisecaver JH, Slot JC, Rokas A. The evolution of fungal metabolic
pathways. PLoS Genet. 2014;10:e1004816.
13
Wong S, Wolfe KH. Birth of a metabolic gene cluster in yeast by
adaptive gene relocation. Nat Genet 2005;37:777–82.
Woodbury W, Spencer AK, Stahmann MA. An improved procedure using ferricyanide for detecting catalase isozymes. Anal
Biochem 1971;44:301–5.
Wysong DR, Christin L, Sugar AM, et al. Cloning and sequencing
of a Candida albicans catalase gene and effects of disruption
of this gene. Infect Immun 1998;66:1953–61.
Yan L, Li M, Cao Y, et al. The alternative oxidase of Candida albicans causes reduced fluconazole susceptibility. J Antimicrob
Chemoth 2009;64:764–73.
Yiannakopoulou EC, Tiligada E. Protective effect of salicylates
against hydrogen peroxide stress in yeast. J Appl Microbiol
2009;106:903–8.