Cytochrome P450 complement (CYPome) of Candida oregonensis, a gut-associated yeast of bark beetle, Dendroctonus rhizophagus

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Cytochrome P450 complement (CYPome) of Candida oregonensis, a gut-associated yeast of bark beetle, Dendroctonus rhizophagus

INEZa, Carlos Iva
n BRIONES-ROBLEROa, Fabiola HERNANDEZ-MART b a, ~ c, Gerardo ZU
NIGA ~ * David R. NELSON , Flor Nohem
ı RIVERA-ORDUNA Departamento de Zoolog
ıa, Escuela Nacional de Ciencias Biolo
gicas, Instituto Polit
ecnico Nacional, Prolongacio
n de
s, M
exico D.F. CP 11340, Mexico Carpio y Plan de Ayala, Col. Sto. Toma b Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 858 Madison Ave. Suite G01, Memphis, TN 38163, USA c Departamento de Microbiolog
ıa, Escuela Nacional de Ciencias Biol
ogicas, Instituto Polit
ecnico Nacional, Prolongaci
on
s, M
exico D.F. CP 11340, Mexico de Carpio y Plan de Ayala, Col. Sto. Toma a

article info

abstract

Article history:

Bark beetles (Curculionidae: Scolytinae) and associated microorganisms must overcome

Received 9 March 2016

a complex tree’s defence system, which includes toxic monoterpenes, to successfully

Received in revised form

complete their life cycle. A number of studies have suggested these microorganisms could

1 June 2016

have ecological roles related with the nutrition, detoxification, and semiochemical pro-

Accepted 8 June 2016

duction. In particular, in filamentous fungi symbionts, cytochrome P450 (CYP) have

Corresponding Editor:

been involved with terpenoid detoxification and biotransformation processes. Candida or-

Richard A. Humber

egonensis has been isolated from the gut, ovaries, and frass of different bark beetle species, and it is a dominant species in the Dendroctonus rhizophagus gut. In this study, we identify,

Keywords:

characterise, and infer the phylogenetic relationships of C. oregonensis CYP genes. The re-

CYP gen

sults indicate that the cytochrome P450 complement (CYPome) is composed of nine genes

Monooxygenases

(CYP51F1, CYP61A1, CYP56D1, CYP52A59, CYP52A60, CYP52A61, CYP52A62, CYP5217A8,

Scolytinae

and CYP5217B1), which might participate in primary metabolic reactions such as sterol

Yeast symbionts

biosynthesis, biodegradation of xenobiotic, and resistance to environmental stress. The prediction of the cellular location suggests that these CYPs to be anchored to the plasma membrane, membranes of the endoplasmic reticulum, mitochondria, and peroxisomes. These findings lay the foundation for future studies about the functional role of P450s, not only for yeasts, but also for the insects with which they interact. ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ52 55 57296000x62418. ~ iga).
n E-mail addresses: [email protected], [email protected] (G. Zu Abbreviations; CYP, cytochrome P450; CPR, cytochrome P450 reductase; SRS, substrate recognition sites; CFEM, commonly found in extracellular membrane proteins; MFS, major facilitator superfamily http://dx.doi.org/10.1016/j.funbio.2016.06.005 1878-6146/ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.


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Introduction Bark beetles are among the most important insect pests of coniferous forests worldwide. They play an important ecological role by promoting natural regeneration and changes in the forest structure (Raffa et al. 2015). However, their outbreaks cause mortality in hundreds of healthy trees over huge areas (Raffa et al. 2008). Bark beetles and their associated microorganisms (e.g., filamentous fungi, bacteria, yeast, and others) must face and overcome a complex tree’s defence system to exploit a specific resource and habitat, the phloem (Six 2013). Specialised chemical compounds (phenolics and terpenoids from oleoresins) and anatomical structures (thick bark, constitutive and traumatic resin ducts, and specialised parenchyma cells) comprise this tree’s defence system (Franceschi et al. 2005; Krokene 2015). Abundant amounts of oleoresin are produced and secreted by trees to resist the attack of insects and their associated microorganisms (Byers 1995; Trapp & Croteau 2001). The flux of constitutive resin released when beetles bore through bark can block the advance of the insects. Additionally, high concentrations of some terpenoids can also damage and kill the beetles and, consequently, their associated microorganisms (Trapp & Croteau 2001; Bakkali et al. 2008; Raffa 2014). A number of studies have documented the diverse functional roles of some of these microorganisms. It is known that filamentous fungi provide nutritional support to bark beetles (Ayres et al. 2000; Bleiker & Six 2007), and some of these fungi are capable to kill trees (Christiansen & Solheim 1990; Harrington 1993), which could facilitate the successful colonisation of a tree (see Six & Wingfield 2011 for another opinion). Fungi also help to overcome the tree’s defence system by metabolising phenolic compounds and terpenoids (Yamaoka et al. 1995; Solheim & Safranyik 1997; Klepzig & Six 2004; DiGuistini et al. 2011; Lah et al. 2013), regulate interactions among themselves and other microorganisms, and affect beetles behaviour by producing pheromonal compounds (Brand et al. 1976; Six 2012; Zhou et al. 2016). In addition to filamentous fungi, bacteria can perform important physiological functions. For example, bacteria are able to degrade complex substrates such as cellulose, xylan, and polymers, which include
nez et al. 2012; Adams starch, lipids, and esters (Morales-Jime et al. 2013; Briones-Roblero et al., unpubl.). Furthermore, bacteria can fix nitrogen and recycle nitrogenous metabolic prod
nez et al. 2009), tolerate and degrade ucts (Morales-Jime monoterpenoids (Adams et al. 2011; Boone et al. 2013; Xu et al. 2016), affect the growth of filamentous fungi, and produce antibiotics against antagonistic fungi (Cardoza et al. 2006; Scott et al. 2008; Adams et al. 2009). Unlike filamentous fungi and bacteria, the functional role of yeasts in the bark beetle life history has been poorly studied, although yeasts are present in all developmental stages from eggs to adult and on the integument, mycangia, intestine, frass, and galleries (Davis 2014). Potential ecological roles have been suggested for yeast isolated from bark beetles, which include nutritional and chemical detoxification, as well as semiochemical emissions (Six 2013; Davis 2014). Over the last several years, different protein-coding genes of many fungi and bacteria, including cytochrome P450

(CYP) monooxygenases, specific monooxygenases and dioxygenases, hydrolases, lyases, and dehydrogenases, and ABC efflux transporters, have been reported to be associated with both terpenoid detoxification and the biotransformation processes of these compounds (Marmulla & Harder 2014 and references therein). Although the ability to detoxify and biotransform these compounds by bark beetle-associated microorganisms has been suggested for a long time, it is only with the development of ‘omics’ technologies and highthroughput sequencing assays that the presence of proteincoding genes that are or may be involved in both processes have been documented (DiGuistini et al. 2011; Adams et al. 2013; Lah et al. 2013; Xu et al. 2016). In particular, CYPs are heme proteins crucial in primary and secondary metabolism pathways and are responsible for most monooxygenation reactions in the phase I metabolism of xenobiotics (Sheehan et al. 2001; Lah et al. 2011; Omiecinski et al. 2011; Aung et al. 2014). CYPs detoxify and/or bioactivate a vast number of xenobiotic chemicals, such as polyaromatic hydrocarbons, via the conversion of lipophilic, nonpolar xenobiotics into more water-soluble, and less toxic metabolites, that are more easily eliminated from the cell (Sheehan et al. 2001; Li et al. 2007; Omiecinski et al. 2011; Kelly & Kelly 2013). Candida oregonensis is a dominant species of yeast that has been isolated from the gut, ovaries, and frass from Dendroctonus and Ips species in all of their developmental stages and in different geographical locations (Shifrine & Phaff 1956; Rivera et al. 2009; Lou et al. 2014). In addition, it has been demonstrated that this yeast species tolerates high concentrations of (þ)-a-pinene (0.200 % v/v), ()-a-pinene (5 % v/v), b-pinene (35 % v/v), and 3-carene (2.300 % v/v). In particular, it has the ability to transform the (þ)-a-pinene into its oxygenated monoterpenes and other products (Briones-Roblero et al., unpubl.). Here, we identify, characterise, and phylogenetically classify C. oregonensis CYPs genes (cytochrome P450 complement e CYPome), that may be involved in the biodegradation of xenobiotic compounds of the host trees and in sterol biosynthesis and resistance to environmental stress.

Materials and methods Insect collection and dissection Emerged adults of Dendroctonus rhizophagus were collected directly from 20 naturally infested Arizona pines, Pinus arizonica Engelm., in San Juanito, Bocoyna Municipality, Chihuahua State (27 550 54.900 N, 107 350 54.600 W), Mexico, in mid-July 2012. From each tree, two beetles (\, _) were taken, because a single pair colonises and kills a tree. All specimens were stored at 4  C in plastic containers with moist paper and transported to the laboratory for further processing. Thirty beetles were superficially disinfected by sequential rinses in sterile distilled water for 1 min; detergent solution (10 mM TriseHCl pH 8, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl; 1 % sodium dodecyl sulfate (SDS); 2 % Triton X-100) for 1 min; 1 % sodium hypochlorite solution for 1 min; 70 % ethanol solution for 1 min; and finally, repeated


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ınez F, et al., Cytochrome P450 complement (CYPome) of Candida oregonensis, Please cite this article in press as: Herna a gut-associated yeast of bark beetle, Dendroctonus rhizophagus, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.06.005

CYPome of Candida oregonensis

washes with sterile distilled water. The last washing water was inoculated in Petri dishes with YPD media (1 % yeast extract, 2 % peptone, and 2 % dextrose, Difco) to assess the efficiency of the disinfections. Plates were incubated at 28  C for 48e72 h. Insects were dissected in a drop of phosphatebuffered solution (PBS, pH 7.400; 137 mM NaCl, 2.700 mM KCl, 10 mM NaHPO4, 2 mM KH2PO4) under sterile conditions using fine-tipped forceps. From each insect, elytra, wings, and tergites were removed, and a longitudinal incision was made on the body to obtain the gut. Then, a set of 30 guts were transferred into a 1.500 ml microfuge tube and homogenised in 1 ml of PBS with sterile pestles, which were used for yeast isolation.

Strain of Candida oregonensis Isolation Candida oregonensis ChDrAdgY58 was isolated from the homogenate by doing 10-fold serial dilutions of the samples and spreading 100 ml of each dilution on plates with YPD medium (supplemented with streptomycin 0.500 mg ml1 and kanamycin 0.500 mg ml1 to inhibit the growth of bacteria) in triplicate. Cultures were incubated at 28  C for 48 h. Based on their morphological characteristics, 60 colonies of yeasts were randomly isolated from the plates. Axenic cultures were stored at 70  C in 50 % glycerol.

Identification DNA extraction was performed from pure colonies of a 24 h culture using the method of Cenis (1992). An ITS region from 300 to 500 bp and another fragment of w600 bp for the 26S rRNA were amplified with the primers ITS1 (50 and ITS4 (50 TCCGTAGGTGAACCTGCGG-30 ) 0 TCCTCCGCTTATTGATATGC-3 ) (White et al. 1990) and with NL1 (50 -GCATATCAATAAGCGGAGGAAAAG-30 ) and NL4 (50 GGTCCGTGTTTCAAGACGG-30 ) (O’Donnell 1993), respectively. PCR amplifications were performed in a thermocycler TC5000 (Techne, Staffordshire, UK) in a 25-ml total reaction volume containing 50e100 ng DNA template, 1 reaction buffer, 2 mM MgCl2, 0.400 mM each primer, 0.400 mM deoxynucleotides (dNTPs), and 1 U recombinant Taq polymerase (Invitrogen Life Technologies, Sao Paulo, Brazil). The reaction conditions were as follows: initial denaturation at 94  C for 5 min, 25 cycles at 94  C for 1 min, annealing temperature 55  C for 1 min, 72  C for 1 min, and final extension at 72  C for 5 min. PCR products were purified with the GeneJET PCR Purification kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and sequenced in an ABI 3130xl Genetic Analyzer (Hitachi, Tokyo, Japan). The taxonomic identification of the yeast was based on the similarity level with respect to reference sequences from the GenBank database. The sequences generated in this study were deposited in the GenBank database under the accession numbers KU144545 for the ITS region and KU144570 for 26S rRNA.

DNA preparation and next-generation sequencing Genomic DNA (gDNA) was extracted from a fresh culture of Candida oregonensis ChDrAdgY58 in YPD broth. The culture

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was harvested in a 50 ml conical centrifuge tube, and the pellet was resuspended in lysis reagent (RiboPure Yeast KitAmbion, Carlsbad, CA, USA). The gDNA was obtained from the yeast suspension following the protocol provided in the RiboPure Yeast Kit (Ambion, Carlsbad, CA, USA) with two modifications: nucleic acid elution was done with 1 TE buffer and an overnight digestion with RNase A 10 mg ml1 (Thermo Scientific, Carlsbad, CA, USA) instead of DNase treatment. The total quantity and quality of gDNA (3.200 mg ml1 in a final volume of 30 ml; 260/280 ¼ 1.800) was measured in a NanoDrop 2000 (Thermofisher Scientific, Wilmington, DE, USA) and submitted to the Otogenetics Corporation (Norcross, GA, USA) for whole genome sequencing. Next, gDNA was subjected to agarose gel and optical density (OD) ratio tests to confirm the purity and concentration prior to fragmentation using Bioruptor Sonicator (Diagenode, Inc., Denville, NJ, USA). Fragmented gDNAs were tested for size distribution and concentration using an Agilent Bioanalyzer 2100 or Tapestation 2200 and NanoDrop 2000. Illumina libraries were made from qualifying fragmented gDNA using an SPRIworks HT Reagent Kit (Beckman Coulter, Inc., Indianapolis, IN, USA), and the resulting libraries were then sequenced on an Illumina HiSeq2000/2500, which generated paired-end reads of 100 nucleotides. Data were analysed for data quality using FASTQC (Babraham Institute, Cambridge, UK). The sequencing data sets (fastq files) were prefiltered with cutadapt to remove sequencing adapters and low-quality reads. The processed paired-end fastq files were input into soapdenovo2 (Luo et al. 2012) for assembling with different Kmer settings from 29 to 100 nt. The assembly with the longest scaffold N50 was selected as the final assembled sequence (Otogenetics Corporation; Norcross, GA, USA).

Data mining CYP genes A Hidden Markov Model-based (HMM) gene structure prediction was performed using the Fgenesh program (Solovyev 2007) in the MolQuest package v 2.4.5.1135 (SoftBerry Inc. 2001e2013). The gene-finding parameters were set specifically for searching in Ascomycetes: Clavispora lusitaniae. Fgenesh retrieved a fasta file with the set of predicted genes and proteins, which was then used to create a protein database with the FormatDB program (MolQuest package) to locally run a Protein Basic Local Alignment Search Tool (BLASTp) analysis.

CYP discoveries in the genome of Candida oregonensis The 4833 genes predicted from the genome of C. oregonensis were mined for CYPs. Full-length CYPs sequences of different families reported for yeast in the GenBank, as well as the conserved regions among cytochromes P450s, which include heme-binding region (FXXGXRXCXG), the PERF domain (PXRX), and the K-helix region (EXXR) (Chen et al. 2014) were used to perform a local BLASTp search (MolQuest package) against the protein database that was previously created. The deduced amino acid sequences identified were submitted to the Cytochrome P450 Nomenclature Committee, and David R. Nelson did the name assignment. All sequences were deposited in GenBank (accession number): CYP52A62


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(KU696336), CYP51F1 (KU696337), CYP61A1 (KU696338), CYP5217A8 (KU696339), CYP52A61 (KU696340), CYP52A60 (KU696341), CYP52A59 (KU696342), CYP56D2 (KU696343), and CYP5217B1 (KU696344).

CYP redox partners search In order to identify the possible cytochrome P450 reductases (CPRs) in the Candida oregonensis genome, the motifs for the Pfam flavin adenine dinucleotide (FAD)-binding domain (Pfam ID: PF00667), the NAD-binding domain (Pfam ID: PF00175), and the Flavodoxin domain (Pfam ID: PF00258) were used. Additionally, we used the cytochrome b5 domain (Pfam ID: PF00173) and the NAD-binding and Oxidoreductase FAD-binding domains (Pfam ID: PF00970A) to identify two possible alternative CYP redox partners: cytochrome b5 and cytochrome b5 reductase. A BLASTp (MolQuest package) search of the reference sequences in the GenBank of the full-length nicotinamide adenine dinucleotide phosphate (NADPH) CPR, cytochrome b5, and cytochrome b5 reductase was done against the protein database that was previously created.

Analysis of full-length CYP sequences The predicted physicochemical characteristics including the molecular mass (kDa) and isoelectric point (IP), of each sequence were determined using the ProtParam program (Gasteiger et al. 2005). Crystal structure data from Saccharomyces cerevisiae CYP51 (Protein Data Bank (PDB) code 4wmz) were used as a template in the ESPript program (Gouet et al. 2003) to assign secondary structure elements onto the corresponding aligned sequences. Substrate recognition sites (SRS) were manually indicated based on the available information of the enzyme CYP51 (Podust et al. 2001; Lepesheva & Waterman 2007). All putatively functional P450 proteins were checked for likely subcellular localisation using the TargetP program (nonplant proteins) (http://www.cbs.dtu.dk/services/TargetP/) (Emanuelsson et al. 2000), WoLF PSORT (fungi) (http://wolfpsort.org/) (Horton et al. 2007), Protcomp-AN v 9.0 (eukaryotic proteins) (MolQuest package), and MitoProt (Claros & Vincens 1996) with default parameters. A multiple protein sequence alignment of all of the complete CYP gene sequences (n ¼ 9) alone or including GenBank reference sequences (n ¼ 79) was done with ClustalX v 2.0.10 (Thompson et al. 1997) using the default parameters. The first

alignment was used to create a protein sequence logo with the web application WebLogo 3.4 (Crooks et al. 2004) to observe sequence conservation and the relative frequency of each amino acid within it. The alignment with the reference sequences was used to perform a phylogenetic inference analysis by maximum likelihood of the full-length CYP sequences with PhyML (Guindon et al., 2010). The best-fit model of protein evolution was selected based on the Akaike information criterion (AIC) in ProtTEST v 2.4 (Abascal et al. 2005). The test supported the LG þ I þ I model with a gamma shape of 1.432 (Le & Gascuel 2008). To estimate the support of each node, a bootstrap was calculated after 1000 pseudoreplicates. The CYP504A8 from Yarrowia lipolytica (XP_504958) was used as outgroup. The phylogenetic tree was deposited in Treebase (https://treebase.org/).

Results Identification of CYP genes A total of 132 sequences were detected as possible CYPs (genome open reading frames e ORFs) based on their similarity to the reference full-length CYPs. A second search was done directed to the motifs of P450 family, which reduced the number of candidates to 25 CYPs. Nine of these CYPs were identified as full-length members of the P450 family, and the remaining CYPs (n ¼ 16) were nonP450 monooxygenases and other enzymes. Three of these, i.e., CYP51F1, CYP56D2, and CYP61A, are previously known members in yeasts; the cytochromes CYP52A59, CYP52A60, CYP52A61, CYP52A62, and CYP5217A8 are new variants within their subfamilies, and CYP5217B1 is the first member of a new CYP5217 subfamily. Each full-length CYP contained the signature CYP motifs (Fig 1).

Identification of CPR A total of 12 sequences were detected as possible NADPH reductase partners. One was identified as a CPR (680 aa), and another was identified as a NADPHeferrihemoprotein reductase (582 aa). Ten more sequences were identified as alternative redox partners; six of them were a fusion mainly of cytochromes b2 and b5. The rest were identified as reduced nicotinamide adenine dinucleotide (NADH)ecytochrome b5 reductase (n ¼ 2) and cytochrome b5 (n ¼ 2).

Fig 1 e Conserved regions in P450s from C. oregonensis. The consensus protein sequence logo (WebLogo 3.4 e Schneider & Stephens 1990) shows the conservation and relative frequency of each amino acid within of highly conserved domains and motifs.


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Table 1 e Physicochemical properties predictive of CYP enzymes encoded by CYP genes from Candida oregonensis. Full-length CYP name

ORF size (bp)

CYP52A59 CYP52A60 CYP52A61 CYP52A62 CYP56D2 CYP51F1 CYP61A1 CYP5217A8 CYP5217B1

1572 1572 1578 1518 1485 1578 1578 1725 1602

M.W. (Da)a 59 59 60 59 57 59 60 65 48

863.800 744.700 704.500 037.100 145.200 593.600 437.900 826.700 219.400

IPa 8.720 8.810 6.940 9.120 9.000 6.410 5.410 7.230 7.970

M.W., Molecular Weight. a As predicted by ProtParam (Gasteiger et al. 2005).

Full-length CYP gene analysis The ORFs of the full-length genes varied from 1485 bp to 1578 bp. All ORFs encoded approximately 500 amino acids. The predicted molecular mass varied from 48 219.4 to 65 826.7 Da, and the IP range from 6.41 to 9.12 (Table 1). The complete amino acid sequences showed the typical conserved P450 domains, including the heme-binding region (FXXGXRXCXG), the PERF domain (PXRX), with the characteristic signature for fungi (PXRW), K-helix region (EXXR), and the oxygen-binding domain (OBD) (AAGXDTT) (Fig 1). The alignment and comparison of the deduced amino acid sequences from Candida oregonensis with respect to the CYP51F1 from Saccharomyces cerevisiae (Fig 2) allowed the identification of the secondary structure of six putative recognition sites (SRSs) that were located in regions with variable structural elements, 9-a helices, and 12-b sheets. The ML-phylogenetic inference analysis (Fig 3) with putative CYP full-length gene sequences showed clustering of the different families in three consistent groups (bootstrap values > 50 %). The first group integrated by two subclusters of the families CYP51 (n ¼ 15) and CYP61 (n ¼ 12), the second group included the CYP52 family from Candida spp. (n ¼ 41), and the third group was formed by two subclusters of families CYP56 (n ¼ 8) and CYP5217 (n ¼ 5). C. oregonensis CYPs clustered within their respective families and showed a major similarity to Clavispora lusitaniae (Table 3). The predictive subcellular localisation of all P450 proteins (Table 2) indicated that cytochromes CYP52A59, CYP52A61, and CYP56D2 might have a glycosidilphosphatidyl inositol (GPI) signal peptide of approximately 20 hydrophobic residues that could anchor the proteins in the endoplasmic reticulum (ER). Because the inferred prediction of cytochromes CYP52A60, CYP52A62, and CYP5217B1 was at the mitochondrial membrane, the MitoProt algorithm was used to define their putative localisation. This test suggested that CYP52A60 had a mitochondrial cleavage sequence in position 70, with a high probability of export to mitochondria. CYP52A62 had the highest probability of export to mitochondria, though, no cleavage signal was found. CYP5217B1 had a mitochondrial cleavage site in position 21 and was predicted as mitochondrial by all of the programs used (Table 2). In contrast,

CYP51F and CYP61A1 were predicted to be bound to plasma membrane and CYP5217A8 to peroxisomal membrane. In addition, CYP52A62 was found in a 13.7 kb cluster (Fig 4A) associated with a gamma-glutamyl transpeptidase, three proteins with a common in fungal extracellular membrane (CFEM) domain, a NADPH ferric reductase, a multidrug transporter of the major facilitator superfamily (MFS), a phosphatidyl-inositol transfer protein, and a zinc fungal transcription factor. Similarly, CYP56D1 was found close to three MFS, one of which was a multidrug efflux permease, and a fungal transcription factor (Fig 4B).

Discussion In this study, we describe the CYPome of Candida oregonensis, which is composed of nine CYPs, i.e., CYP51F1, CYP61A1, CYP56D2, CYP52A59, CYP52A60, CYP52A61, CYP52A62, CYP5217AB, and CYP5217B1, from five different families. The number of CYPs genes (3e12) known that compose the differents CYPome of yeasts is smaller than filamentous fungi (28e153) (Chen et al. 2014). Our results show that the C. oregonensis CYPome has a number of CYPs within this range. This CYPs number is similar to those reported for other yeast species such as Clavispora lusitaniae, Candida albicans, Debaryomyces hansenii, and Meyerozyma guilliermondii (Table 4) (Nelson 2009; Moktali et al. 2012; Chen et al. 2014). The phylogenetic analysis shows that C. oregonensis CYPs are more related to C. lusitaniae, which is not surprising as C. oregonensis belongs to the Clavispora/Candida clade of the Metschnikowiaceae family where C. lusitaniae is contained. The families CYP51F, CYP56D, CYP61A, CYP52A, and CYP5217 are present in both yeasts (Nelson 2009), and CYP501 only is present in C. lusitaniae.

CYPs of primary metabolism in Candida oregonensis The identification of the genes CYP51F1, CYP56D2, and CYP61A1 in C. oregonensis suggests that CYP enzymes can play important roles in the primary metabolism of yeasts. Several studies have documented that the families CYP51 and CYP61 in other species of yeast as Saccharomyces cerevisiae and Candida albicans, have housekeeping functions in sterol  2011; Moktali biosynthesis (Kelly et al. 2009; Cresnar & Petric et al. 2012; Chen et al. 2014). The CYP56 family has been characterised in both S. cerevisiae and C. albicans (Melo et al. 2008;  2011). CYP56 (called DIT2) in these species is Cresnar & Petric involved in meiotic spore wall biogenesis, particularly in dityrosine biosynthesis (Briza et al. 1994; Melo et al. 2008; Cresnar &  2011). These same studies reported that the DIT2 ORF is Petric adjacent to the DIT1 gene, which is not a CYP related to spore wall maturation (Briza et al. 1994; Melo et al. 2008). The DIT1 gene in C. oregonensis (pos. 6 117 912e6 119 324) is located at z 4.700 kb from CYP56-DIT2 (pos. 1 342 120e1 343 604). It is flanked upstream by a fungal transcription factor and a MFS transporter and downstream by two MFS transporters (Fig 4B). The closest MFS is 60 bp downstream. Though the MFS transporters were not fully characterised, the one flanking the CYP upstream (Fig 4B) appears to be part of the MFS DHA1 subfamily. Members of the DHA1 subfamily,


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Fig 2 e Multiple sequence alignment and assignment of the secondary structural elements. The alignment included all cytochromes P450s from C. oregonensis, and the predicted S. cerevisiae CYP51 protein sequence (PDB code 4wmz). SRS 1e6 as well as motifs (HKM e helix K motif and HBM e heme-binding motif) and conserved regions (OBD, PERF e PERF domain) in the alignment were manually determined.


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CYPome of Candida oregonensis

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Fig 2 e (continued).

such as Dtr1, facilitate the translocation of dityrosine through the prospore membrane during spore wall maturation (Felder et al. 2002; Nickas et al. 2003). Qdr1, another member of the DHA1 subfamily, participates in the assembly of the spore

wall (Felder et al. 2002; Lin et al. 2013; Dos Santos et al. 2014). This evidence suggests that these genes are associated in the same pathway. Further studies must be done to characterise both the MFS and CYP function.


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Fig 3 e Maximum-likelihood tree of CYP based on amino acid sequences from C. oregonensis plus GenBank sequences. The analysis was performed using the amino acid substitution model LG D G D I with a gamma parameter of 1.432. The accession numbers of GenBank sequences are shown in brackets. Bootstrap values after 1000 pseudoreplicates are shown at nodes. *Sequences were obtained from CYP homepage (Nelson 2009).

The CYP52 family was the most abundant in C. oregonensis. A number of studies have related members of this family to alkane metabolism (Craft et al. 2003; Van Bogaert et al. 2011; Huang et al. 2014), multidrug resistance (Kim et al. 2007), and of metabolism of xenobiotics through the b-oxidation pathway (Chen et al. 2014). The CYP52 family has several isoforms that exhibit diverse activities towards longchain hydrocarbons, fatty acids or molecules with similar structures (Van Bogaert et al. 2011). For example, it has

been demonstrated in Candida maltosa that CYP52A3 converts hexadecane to hexadecanol (Scheller et al. 1998), and CYP52A21 from C. albicans hydroxylates lauric, myristic, and palmitic acids. In addition, CYP52A21 confers multidrug resistance to this last species (Kim et al. 2007). Gas chromatographyemass spectrometry (GCeMS) assays in C. oregonensis show that this yeast biotransforms the monoterpene (R)(þ)-a-pinene to trans-verbenol (Briones-Roblero et al., unpubl.).


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ınez F, et al., Cytochrome P450 complement (CYPome) of Candida oregonensis, Please cite this article in press as: Herna a gut-associated yeast of bark beetle, Dendroctonus rhizophagus, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.06.005

CYPome of Candida oregonensis

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Table 2 e Subcellular localisation of predictive P450 enzymes of Candida oregonensis using different softwares. Full-length CYP name CYP52A59

CYP52A60

CYP52A61

CYP62A62

CYP56D2

CYP51F1

CYP61A1

CYP5217A8

CYP5217B1

WoLF PSORT (Horton et al. 2007)

Protcomp-AN (SoftBerry Inc.)

TargetP (Emanuelsson et al. 2000)

MITOPROT (Claros & Vincens 1996)

Cyto 8.500 Mito 5 ER 4 Mito 11 Cyto 5 Nucl 3 Cyto 10.500 ER 6 Mito 2 Mito 6 Cyto 5.500 ER 3 Nucl 7 Cyto 5 ER 6 Cyto 9.500 ER 8 Nucl 7 Cyto 11 Cyto 9 Peroxi 8.300 Cyto 10.500 Nucl 7 ER 6 Mito 10 Plas 7 Cyto 5

MB-ER GPI-signal 503 Score 7.990 MB-ER GPI-signal 503 Score 7.400 MB-ER GPI-signal 488 Score 7.100 MB-ER GPI-signal 472 Score 7.100 MB-ER GPI-signal 480 Score 6.300 Plasmatic membrane GPI-signal 489 Score 9.200 Plasmatic membrane Score 4.400

SP 0.923

PE ¼ 0.673

SP 0.934

PE ¼ 0.771 Cleavage site ¼ 70

SP 0.522

PE ¼ 0.055

SP 0.778

PE ¼ 0.876

SP 0.975

PE ¼ 0.024

SP 0.843

PE ¼ 0.019

Other 0.662

PE ¼ 0.149

Membrane-bound peroxisomal Score 8.500 Mitochondria Score 5.700

SP 0.983

PE ¼ 0.051

Mit 5.700

PE ¼ 0.174 Cleavage site ¼ 21

Cyto: cytoplasmic; Mito: mitochondrial; Nucl: nuclear, Peroxi: peroxisomal, Plas: plasmatic membrane. MB-ER: membrane-bound endoplasmic reticulum; GPI: Glycosilphosphatidyl inositol. SP: secretory pathway; Mit: mitochondria. PE: possibility of export to mitochondria.

The presence of members of the CYP52 family in C. oregonensis might be explained by its ability to transform the monoterpenes present in the bark beetle gut. These compounds are toxic to microorganisms as well as the beetle. These compounds affect the yeast cell membrane permeability and fluidity, disrupt the cytoplasmic membrane, and inhibit the respiratory chain in mitochondria (Uribe et al. 1990; Parveen et al. 2004; Bakkali et al. 2008; Brennan et al. 2013; Mart
ınez et al. 2014). In bark beetles, it is known that the monoterpenes a-pinene, b-pinene, and 3-carene can damage the membranes
pez et al. 2011). of midgut epithelial cells and mitochondria (Lo

Finally, the functional role of CYP5217 family has not been characterised, though it has been detected in C. albicans, Clavispora lusitaniae, and Candida dubliniensis (Nelson 2009; Chen et al. 2014).

CYPs putative localisation in Candida oregonensis Class II cytochromes P450s are the most common in eukaryotic organisms, and are usually localised in the ER (Cresnar  2011). The in silico analysis of the subcellular location & Petric of CYP52A59, CYP52A61, and CYP56D2 genes in this study

Fig 4 e Flanking regions CYP52A62 and CYP56D2 (DIT2) regions from C. oregonensis. Panel A. An overview of the gene cluster contiguous CYP52A62. Panel B. The CYP56D2-DIT2 gene located between MFSs; the DIT1 gene is found at 4.7 kb upstream.
ndez-Mart
ınez F, et al., Cytochrome P450 complement (CYPome) of Candida oregonensis, Please cite this article in press as: Herna a gut-associated yeast of bark beetle, Dendroctonus rhizophagus, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.06.005


ndez-Mart
ınez et al. F. Herna

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Table 3 e Aminoacidic similarity percentage of putative CYP from Candida oregonensis with CYP sequences in yeasts. C. oregonensis CYP CYP51F1 CYP56D2 CYP61A1 CYP52A59 CYP52A60 CYP52A61 CYP52A62 CYP5217A8 CYP5217B1

Reference CYPa

Organism

Similarity (%)

CYP51F1 CYP56D1 CYP61A1 CYP52A40 CYP52A29 CYP52A30 CYP52A31 CYP5217A4 CYP5217A7

Clavispora lusitaniae C. lusitaniae C. lusitaniae Pichia guilliermondii C. lusitaniae C. lusitaniae C. lusitaniae P. guilliermondii Scheffersomyces stipitis

79 53 80 61 63 66 56 56 45

a Sequences obtained from CYP homepage (Nelson 2009).

suggests that they are bound to the ER membrane. The rest of the cytochromes P450s of C. oregonensis were predicted to have a different localisation: CYP51F and CYP61A1 in plasma membrane, CYP5217A8 in peroxisomal membrane, and CYP5217B1 in mitochondria. Nonetheless, in situ studies must be done to corroborate the predictive localisations of the genes. It is known that proteins such as CYP2C2 have retention signals in the region of its transmembranal sequence that make them resident ER proteins (Szczesna-Skorupa et al. 2003). However, when such a signal is lacking, proteins are transported to other regions within the cell (SzczesnaSkorupa & Kemper 2001), as is the case for the human CYP51A1 (Cotman et al. 2004). Therefore, if cytochromes P450s are present in plasma membranes of C. oregonensis, they may play an important role in membrane fluidity and permeability regulation (Lepesheva & Waterman 2007). CYPs located in peroxisomes may play a role in the metabolism of alcohol, biosynthesis of cholesterol, hydroxylation of fatty acids, and xenobiotic compounds (Pahan et al. 1997). Evidence in previous studies suggests mitochondrial P450 cytochromes can be stimulated by the same compounds as ER P450s (Niranjan et al. 1984, 1985; Anandatheerthavarada et al. 1997; Jung & Di Giulio 2010); however, further studies must be done to understand the role of mitochondrial P450s.

Table 4 e Putative P450 number and family in some members of Ascomycota. Yeast species

CYP number in genome

Number of family type

9 10 3 12 8 9 5 10 9 5 3 10 17 9

6 6 3 6 6 5 5 5 6 5 3 6 6 5

Candida albicans Candida dubliniensis Candida glabrata Candida tropicalis Clavispora lusitaniae Debaryomyces hansenii Kluyveromyces lactis Lodderomyces elongisporus Meyerozyma guilliermondii Ogataea parapolymorpha Saccharomyces cerevisiae Scheffersomyces stipitis Yarrowia lipolytica Candida oregonensis Modified from Chen et al. (2014).

The putative localisations of CYP52A60 and CYP52A62 are uncertain, because the in silico analysis using different software indicated that they might be located both in membrane of the ER or in mitochondria. A similar situation has been described for CYP1A in mummichog fish (Fundulus heteroclitus), which was expressed both in ER and mitochondria (Jung & Di Giulio 2010).

CYP52A62 cluster in Candida oregonensis Fungi, particularly filamentous fungi, produce a wide variety of secondary metabolites that even though these are not essential; however, they can provide protection against various environmental stresses (Khaldi et al. 2010; Lah et al. 2011). The genes that are responsible for their biosynthesis, export, and transcriptional regulation are often found in contiguous gene clusters in the genome (Kelly et al. 2009; Khaldi et al. 2010; Lah et al. 2013). The cluster identified in C. oregonensis includes an exporter protein (MFS), a transcription regulator (zinc fungal transcription factor), a modifying enzyme (CYP52A62), a specific ferric reductase coupled to CYP52A62, and three CFEM domains. In filamentous fungi, these clusters are integrated in the same way (Kelly et al. 2009; Khaldi et al. 2010; Lah et al. 2013), plus a ‘backbone’ enzyme (polyketide synthase e PKS or a nonribosomal peptide synthase eNRPS), which is not present in C. oregonensis. The absence of this enzyme has also been reported in Saccharomyces cerevisiae (Khaldi et al. 2010). A similar cluster was found in Grosmannia clavigera, a fungal associate to the mountain pine beetle (Dendroctonus ponderosae), that contains a CYP630B18, a ferric reductase (CPR2), a transporter protein, and a specific fungal transcription factor, all of which were induced following lodge pole extract treatment (DiGuistini et al. 2011; Lah et al. 2013). Studies of functional expression of the complete cluster of C. oregonensis are necessary to know whether this cluster has a similar function as in G. clavigera. The function of the three CFEM domains present in the cluster of C. oregonensis is unknown. Nevertheless, in nonpathogenic fungi as S. cerevisiae and Aspergillus fumigatus, it has been reported that these domains are involved in cell wall biogenesis and play an important role in maintaining the integrity and stability of the cell (Moukadiri et al. 1997; Vaknin et al. 2014).

Conclusions The present study reveals that Candida oregonensis, which is a gut symbiont of Dendroctonus rhizophagus, possesses


ndez-Mart
ınez F, et al., Cytochrome P450 complement (CYPome) of Candida oregonensis, Please cite this article in press as: Herna a gut-associated yeast of bark beetle, Dendroctonus rhizophagus, Fungal Biology (2016), http://dx.doi.org/10.1016/ j.funbio.2016.06.005

CYPome of Candida oregonensis

different CYPs that are involved in essential functions for cell survival such as sterol biosynthesis and resistance to adverse environmental conditions. Moreover, the yeast CYPome, particularly the CYP52 family, might aid in the detoxification of the terpenoid compounds during the host tree colonisation and life cycle of bark beetles. The presence of a similar cluster, such as that reported in Grosmannia clavigera, might confer a fitness advantage that can benefit not only the yeast but also the bark beetle. Finally, these findings lay the foundation for future experiments to elucidate the role of P450s in the symbioses between yeastbark beetles.

Acknowledgements
n Molina, Salvador Embarcadero We thank Gabriel Obrego
nez, Rosa Mar
ıa Pineda Mendoza, and two anonymous reJime viewers for their valuable comments, suggestions, and observations. This investigation was supported by Instituto de Ciencia y Tecnolog
ıa del Distrito Federal 45/2012 and SEPeCONACYT 169494. This work was part of F.H.M.’s PhD dissertation. F.H.M. (246641) and C.I.B.R. (227280) were CONACYT fellows.

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