RESEARCH ARTICLE Open Access

Genome sequence of the filamentous soilfungus Chaetomium cochliodes revealsabundance of genes for heme enzymesfrom all peroxidase and catalasesuperfamiliesMarcel Zámocký1,2*, Hakim Tafer3, Katarína Chovanová2, Ksenija Lopandic3, Anna Kamlárová2

and Christian Obinger1

Abstract

Background: The ascomycetous family Chaetomiaceae (class Sordariomycetes) includes numerous soilborn,saprophytic, endophytic and pathogenic fungi which can adapt to various growth conditions and living niches byproviding a broad armory of oxidative and antioxidant enzymes.

Results: We release the 34.7 Mbp draft genome of Chaetomium cochliodes CCM F-232 consisting of 6036 contigswith an average size of 5756 bp and reconstructed its phylogeny. We show that this filamentous fungus is closelyrelated but not identical to Chaetomium globosum and Chaetomium elatum. We screened and critically analysedthis genome for open reading frames coding for essential antioxidant enzymes. It is demonstrated that the genomeof C. cochliodes contains genes encoding putative enzymes from all four known heme peroxidase superfamiliesincluding bifunctional catalase-peroxidase (KatG), cytochrome c peroxidase (CcP), manganese peroxidase, twoparalogs of hybrid B peroxidases (HyBpox), cyclooxygenase, linoleate diol synthase, dye-decolorizing peroxidase(DyP) of type B and three paralogs of heme thiolate peroxidases. Both KatG and DyP-type B are shown to beintroduced into ascomycetes genomes by horizontal gene transfer from various bacteria. In addition, two putativelarge subunit secretory and two small-subunit typical catalases are found in C. cochliodes. We support our genomicfindings with quantitative transcription analysis of nine peroxidase & catalase genes.

Conclusions: We delineate molecular phylogeny of five distinct gene superfamilies coding for essential hemeoxidoreductases in Chaetomia and from the transcription analysis the role of this antioxidant enzymatic armory forthe survival of a peculiar soil ascomycete in various harsh environments.

Keywords: Chaetomium cochliodes, Peroxidase-catalase superfamily, Peroxidase-cyclooxygenase superfamily,Peroxidase-chlorite dismutase superfamily, Peroxidase-peroxygenase superfamily, Heme-catalase super family

* Correspondence: marcel.zamocky@boku.ac.at1Department of Chemistry, Division of Biochemistry, University of NaturalResources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria2Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta21, SK-84551 Bratislava, SlovakiaFull list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Zámocký et al. BMC Genomics (2016) 17:763 DOI 10.1186/s12864-016-3111-6

BackgroundThe ascomycetous family of Chaetomiaceae (class Sor-dariomycetes) includes numerous soilborn, saprotrophic,endophytic and pathogenic fungi that apparently exhibita large flexibility in their adaptation to various growthconditions and living niches. In Mycobank (www.myco-bank.org) currently up to 451 members of this abundantfungal family are registered but only from two represen-tatives (i.e. Chaetomium thermophilum and Chaetomiumglobosum) the completely sequenced genomes are avail-able. Analysis of the genome of C. thermophilum [1]mainly focused on the presence of genes coding fornucleoporins of high thermal stability, whereas the draftgenome of Chaetomium globosum [2] was mainly askedfor diverse genes coding cellulolytic pathways.The filamentous fungus Chaetomium cochliodes was

long considered to be a variant of Chaetomium globosum(cf. the NCBI taxonomy database at www.ncbi.nlm.nih.-gov/taxonomy) but already in very early studies e.g. [3] itwas shown that C. cochliodes produces the antibioticchaetomin which was shown to be highly active mainlyagainst Gram-positive bacteria. Additionally, studiesfrom our laboratory revealed differences between C. glo-bosum and C. cochliodes in the primary sequence andexpression profile of peroxisomal catalase-peroxidases[4]. These findings – together with the fact that peroxi-dases participate in diverse fungal secondary metabolismpathways [5–9] – prompted us to sequence the entiregenome of Chaetomium cochliodes strain CCM-F232 fordetailed comparative studies.Here we release the draft genome of C. cochliodes, re-

construct its phylogeny and analyse the occurrence ofabundant genes coding for heme containing peroxidasesand catalases with respect to the recently described fourdistinct heme peroxidase superfamilies [10] and theheme catalase super family [11]. Interestingly, represen-tatives from all five (super)families were found includingputative bifunctional catalase-peroxidase, cytochrome cperoxidase, hybrid B peroxidases, cyclooxygenase-likeenzymes, dye-decolorizing peroxidases, heme thiolateperoxidases as well as large- and small-subunit mono-functional catalases. The occurrence of this large num-ber and variability of genes encoding hemehydroperoxidases in C. cochliodes is discussed in com-parison with related fungal genomes. We support ourgenomic findings with a first round of a quantitative ex-pression analysis of selected genes from all mentionedsuperfamilies involved in the catabolism of H2O2.

MethodsSource and cultivation of Chaetomium cochliodes andisolation of genomic DNAChaetomium cochliodes CCM F-232 was obtained fromCzech Collection of Microorganisms at the Masaryk

University, Faculty of Natural Sciences in Brno, CzechRepublic. The composition of the incubation mediumand the growth conditions were the same as describedpreviously [4].Genomic DNA from 100 mg of frozen fungal myce-

lium was isolated with the method of Carlson [12] byusing 2 % CTAB in a modification suitable for genomesequencing described in [13]. Finally, extracted DNAwas completely dissolved in TE buffer (10 mM Tris–HCl 1 mM EDTA, pH 8.0) to a final volume of 100 μL.The concentration of obtained sample was measured inNanodrop 2000 (Thermo Scientific, Waltham, MA,USA).

Library preparation for DNA sequencingApproximately 1 μg of high quality genomic DNA wasfragmented in 50 μl Low TE buffer (10 mM Tris pH 8.0,0.1 mM EDTA) by BioRuptor UCD-200 sonication sys-tem (Life Technologies, Carlsbad, CA, USA) to obtain apopulation of ~190 bp long fragments. The length andthe quantity of generated fragments were assessed byBioanalyzer chip technology (Agilent Technologies,Santa Clara, CA, USA) according to the manufacturer’sinstructions. The protocol of the Library Builder™ Sys-tem (Life Technologies, Carlsbad, CA, USA) was usedfor adaptor ligation, nick repair and fragment purifica-tion. The selection of 270 bp long fragments was con-ducted by the Pippin Prep instrument (Sage Science,Beverly, MA, USA) according to the manufacturer’s in-structions. Library quantification was carried out usingthe TaqMan qPCR protocol of Life Technologies.

Genomic DNA sequencing and ORF predictionWhole genome sequencing was carried out using theIon Proton Technology (including the Ion AmpliSeq li-brary preparation kit, Template OT2 200 kit, Ion PI se-quencing 200 kit, and the Ion PI chip kit version 2; LifeTechnologies, Carlsbad, CA), according to the instruc-tions of the manufacturer. A total of 34.746 Mbp, with amedian read length of 180 bp, were assembled into a draftgenome containing 6036 contigs (N50, 14,381). The gen-ome assembly was performed with Newbler 2.9. Genomecoverage of this sequencing was 316 x. The entire genomeshotgun project was deposited at GenBank under acces-sion LSBY00000000, BioProject PRJNA309375, BioSampleSAMN04432217. For comparative genomic analyses ofChaetomium cochliodes genes Ensembl Fungi (http://fun-gi.ensembl.org/index.html) was used.For gene prediction in sequenced C. cochliodes con-

tigs, HMM-based methods FGENESH and FGENESH+ located at www.softberry.com [14] trained forclosely related C. globosum & C. thermophilum wereused. For all peroxidase and catalase genes they werealso curated manually.

Zámocký et al. BMC Genomics (2016) 17:763 Page 2 of 15

Reconstruction of fungal phylogenySelected DNA sequence spanning the region from the 3′end of the 18S rDNA, the complete ITS1, 5.8S rDNA,ITS2 and the 5′ end of the 28S rDNA from correspond-ing C. cochliodes contigs was aligned with 33 related se-quences from Ascomycetes in exactly the same regionobtained from GenBank (Table 1). This DNA alignmentwas performed with the Muscle program [15] imple-mented in MEGA 6 package with its default parametersand 100 iterations. For subsequent phylogeny

reconstruction MEGA 6 program suite [16] was appliedon this 2474 bp long DNA alignment containing the typ-ical fungal barcode motif [17]. Maximum likelihoodmethod with 1000 bootstrap replications and generaltime reversed substitution model were applied. Further,uniform rates of substitutions with invariant sites andinvolvement of all aligned sites with nearest-neighbourinterchange and very strong branch swap filter were se-lected as optimised parameters. The resulting tree wasrendered with Tree Explorer of the same MEGA

Table 1 List of all DNA sequences with their GenBank accession numbers used for phylogeny reconstruction in the region 18S, ITS1,5.8S, ITS2, 28S-rDNA

Abbrev. Fungus Taxonom. family GB accession # [bp] used for phyl.

Ccoch Chaetomium cochliodes Chaetomiaceae KT895345 2217

Celat Chaetomium elatum Chaetomiaceae M83257 2211

Cg1 Chaetomium globosum CBS148.15 Chaetomiaceae NT_166001 2245

Cg2 Chaetomium globosum (endophyt) Chaetomiaceae DQ234257 2219

Cg3 Chaetomium globosum isol. W7 Chaetomiaceae JQ686920 2219

Cthe Chaetomium thermophilum Chaetomiaceae GCA_000221225 2237

Coacu1 Colletotrichum acutatum 1 Glomerellaceae AJ301905 2227

Coacu2 Colletotrichum acutatum 2 Glomerellaceae AJ301906 2225

Cocir Colletotrichum circinans Glomerellaceae AJ301955 2216

Cococ Colletotrichum coccodes Glomerellaceae AJ301957 2218

Codem Colletotrichum dematium Glomerellaceae AJ301954 2220

Colup Colletotrichum lupini Glomerellaceae AJ301959 2200

Cotri Colletotrichum trifolii Glomerellaceae AJ301942 2231

Cotru Colletotricum truncatum Glomerellaceae AJ301937 2213

Fgram Fusarium graminearum Nectriaceae NC_026477 2188

Gcin Glomerella cingulata Glomerellaceae AJ301952 2198

Hgri Humicola grisea Chaetomiaceae AY706334 2202

Lsak Lecanicillium saksenae Cordycipitaceae AB360363 2236

Masp Madurella sp. TMMU3956 Sordariaceae EU815932 2271

Mhin Myceliophthora hinnulea Chaetomiaceae JQ067909 2099

Mthe Myceliophthora thermophila Chaetomiaceae NC_016478 2217

Mgram Mycosphaerella graminicola Mycosphaerellaceae NC_018212 2195

Matr Myrothecium atroviride Stachybotriaceae AJ302002 2223

Mcin1 Myrothecium cinctum 1 Stachybotriaceae AJ301996 2204

Mcin2 Myrothecium cinctum 2 Stachybotriaceae AJ302004 2202

Mver Myrothecium verrucaria Stachybotriaceae AJ301999 2222

Ncr Neurospora crassa Sordariaceae FJ360521 2230

Pan Podospora anserina Lasiophaeriaceae FO904938 2196

Sfim Sordaria fimicola Sordariaceae X69851 2256

Taus Thielavia australiensis Chaetomiaceae JQ067908 2160

Tter Thielavia terrestris Chaetomiaceae NC_016459 2233

Tasp Trichocladium asperum Chaetomiaceae AY706336 2202

Tatr Trichoderma atroviride Hypocreacea NW_014013638 2251

Vcil Volutella ciliata Nectriaceae AJ301967 2214

Zámocký et al. BMC Genomics (2016) 17:763 Page 3 of 15

package. For additional verification, the same 2474 bplong DNA alignment was subjected to phylogeny re-construction using MrBayes 3.2 [18]. Majority consen-sus tree was obtained from all credible topologiessampled by MrBayes over 200,000 generations (with astandard deviation of split frequencies below 0.01) byusing the same GTR substitution model with gammadistributed rate variation across sites and a proportionof invariable sites.

Reconstruction of molecular phylogeny of proteinsuperfamiliesSelected protein sequences translated from C. cochliodescontigs (Table 2B) and similar protein sequences codingfor various peroxidases and catalases (i.e. hydroperoxi-dases deposited at PeroxiBase http://peroxibase.toulou-se.inra.fr with direct links to GenBank & UniProt) werealigned with the Muscle program [15] using default pa-rameters and 100 iterations. Obtained alignments wereinspected and ambiguously aligned regions were ex-cluded from further analysis. Resulting alignments weresubjected to protein phylogeny reconstruction usingMEGA 6 [16] with optimized parameters according tolowest Bayesian information criterion scores (Additionalfile 1: Table S1). Maximum likelihood method with 100bootstraps was chosen using the best substitution modelfor each alignment (WAG in three cases and LG in twocases cf. Additional file 1: Table S1 for details), gammadistribution of rates (four categories) and the presenceof invariant sites. Nearest-neighbour interchange wasused as heuristic method and very strong branch swapfilter was applied. The same protein alignments weresubjected to phylogeny reconstruction using MrBayes3.2 [18]. Majority consensus tree was obtained from allcredible topologies sampled by MrBayes over 500,000generations (with a standard deviation of split frequen-cies below 0.10) by using the same substitution model asin MEGA. Resulting trees were rendered with FigTreegraphic suite available at http://tree.bio.ed.ac.uk/soft-ware/figtree as cladograms with transformed branches.

Transcriptional analysis of genes involved in peroxidecatabolism with RT-qPCRTo study the level of expression of genes involved inperoxide catabolism either non-induced C. cochliodessamples or samples induced in the early exponentialphase of growth with 5 mM H2O2 or 5 mM PAA (finalconcentration, added only for last 30 min.) were used fortotal RNA isolation with RNeasy Plus Mini kit (Qiagen,Netherlands). Obtained RNA samples were directly sub-jected to RT-qPCR assays in AriaMx6 device (AgilentTechnologies, Sancta Clara CA, USA) using the BrilliantIII Ultra Fast SYBR Green Master Mix (also from

Agilent Technologies) with specific primers for selectedgenes listed in Table 3.

Results and discussionOverview of the sequenced genome of Chaetomiumcochliodes CCM F-232In total 6036 contigs were obtained from the genomicDNA of C. cochliodes strain CCM F-232 deposited atGenBank under accession LSBY00000000, BioProjectPRJNA309375, BioSample SAMN04432217. 4141 ofthese contigs were larger than 500 bp. The genome sizeof the complete assembly was determined to be34,745,808 bp. This value is very near to previously de-termined size of closely related C. globosum (updated to34.9 Mb) [2]. The GC content of the entire genome ofC. cochliodes was estimated to 55.95 % which is a slightdifference to the corresponding value for C. globosum(55.6 %). The average size of C. cochliodes large genomiccontigs (>500 bp) in this experiment was determined as8256 bp, the N50 contig size was 14,381 bp and the lar-gest assembled contig comprised 109,425 bp. As a qual-ity control Phred quality scores were determinedaccording to Illumina device: the portion of Q40+ baseswas 34,112,976 (99.83 % of the whole genome sequencedraft) whereas Q39− bases portion was only 59,430(0.17 %). Prediction of all possible ORFs of C. cochliodeswith Chaetomia-optimised FGENESH suite [14] led inboth DNA strands to a total value of 10,103. This countis lower than the estimation for mesophilic C. globosum[2] but much higher than the estimation for C. thermo-philum [1] or related thermophilic fungi. A brief com-parison of three related fungal genomes is presented inTable 4. The average count of exons per predicted C.cochliodes gene was calculated as 3 with FGENESH.

Phylogeny reconstruction in the 18S r DNA – ITS1 – 5.8S rDNA – ITS2 – 28S r DNA regionFirst, we were interested in the exact phylogenetic pos-ition of Chaetomium cochliodes. For this purpose wehave reconstructed the DNA phylogeny of its 2217 bpregion spanning the region from the 3′ end of the 18SrDNA, the complete ITS1, 5.8S rDNA, ITS2 and the 5′end of the 28S rDNA containing the highly conservedlocus described as universal fungal barcode [17]. Besidesall corresponding DNA sequences for species of theChaetomiaceae family currently available in GenBank,also sequences from related ascomycetous families wereincluded in this reconstruction (Table 1). The DNA align-ment used for the phylogeny reconstruction (Additional file2: Figure S1) reveals clear differences (i.e. substitutions, in-sertions and deletions) in the sequence of C. cochliodes ifcompared with corresponding sequences of C. globosum inthe entire region. The phylogenetic output presented inFig. 1 (obtained by two independent methods) clearly

Zámocký et al. BMC Genomics (2016) 17:763 Page 4 of 15

segregates Chaetomium cochliodes from closely related C.elatum which is a root-colonizing fungus whose genome isnot yet sequenced [19]. Both these fungi are separated froma sister clade represented by three different DNA sequenceswithin this region coding for various C. globosum strainswith a high statistical support. This figure clearly

demonstrates that the thermophilic representatives (mainlyC. thermophilum but also e.g. T. terrestris and M.thermo-phila) of the Chaetomiaceae family can be considered asbasal lineages of the Chaetomia clade thus suggesting thatmesophily has evolved only secondarily in this lineage. Ourresults correlate with the previous work on thermophilic

Table 2 List of potentially all genes coding for enzymes involved in H2O2 metabolism in contigs of C. cochliodes genome

Gene name In contig # Seq.identity*

Closestneighbour**

#Introns

Gene-superfamily relations

A) genes coding for enzymes producing H2O2

CcochCuZnSOD 0613 98 % CgCuZnSOD 4 Copper/zinc superoxide dismutase superfamily (SODC)

CcochDAAO 0702 85 % Mth_G2QLH3 3 Flavin D-amino acid oxidase (peroxisomal)

CcochFeMnSOD1 0353 91 % TthFeMnSOD 2 Iron/manganese superoxide dismutase superfamily

CcochFeMnSOD2 1984 &0805

93 % CgFeMnSOD1 3 Iron/manganese superoxide dismutase superfamily

CcochFeMnSOD3 0879 94 % CgFeMnSOD2 1 Iron/manganese superoxide dismutase superfamily

CcochFlOx1 0556 55 % Colgra_E3Q5F0 1 GMC superfamily (flavin oxidases)

CcochGlOx1 0600 53 % Scap_A0A084G823 7 GMC superfamily (flavin oxidases): glucose oxidase

CcochNOx1 0029 93 % CgNox2 2 NADPH oxidase

B) genes coding for enzymes degrading H2O2

CcochkatG1 0012 93 % CgkatG1 none peroxidase-catalase superfamily: bifunctional catalase-peroxidase

Ccochccp 0676 95 % Cgccp 2 peroxidase-catalase superfamily: cytochrome c peroxidase

Ccochpox2a 3115 &3438

68 % Cthepox2a 1 peroxidase-catalase superfamily: Family II prob. manganese-dependent

CcochhyBpox1 3712 &3350

100 % CghyBpox1 none peroxidase-catalase superfamily: hybrid B peroxidase

CcochhyBpox2 0794 93 % CghyBpox2 1 peroxidase-catalase superfamily: hybrid B peroxidase

Ccochcyox1 2133 &0418

83 % CgCyOx1 3 peroxidase-cyclooxygenase superfamily: cyclooxygenase

Ccochlds 1074 &4463

91 % Cglds1 5 peroxidase-cyclooxygenase superfamily: linoleate diol synthase

Ccochdyprx 0391 89 % Cgdyprx none peroxidase-dismutase superfamily: Dyp_B peroxidase (fusion w. PFL)

Ccochhtp1 1650 92 % Cghtp1 3 peroxidase-peroxygenase superfamily: heme-thiolate peroxidase

Ccochhtp2 2302 95 % Cghtp3 3 peroxidase-peroxygenase superfamily: heme-thiolate peroxidase

Ccochhtp3 1018 85 % Cghtp4 2 peroxidase-peroxygenase superfamily: heme-thiolate peroxidase

Ccochvcpo 0469 &1446

93 % Cgvcpo 3 non heme peroxidases: vanadium haloperoxidase

Ccochgpx 0466 84 % Mthgpx 1 non-metal peroxidases: glutathione peroxidase

Ccoch1cysprx 1586 96 % Cg1cysprx 1 non metal peroxidases: 1-cysteine peroxiredoxin

Ccoch2cysprx 1595 99 % Cg2cysprx 2 non-metal peroxidases: typical 2-cysteine peroxiredoxin

CcochprxII 0388 &1977

95 % CgprxII 1 non-metal peroxidases: atypical 2-cysteine peroxiredoxin

CcochkatA1 0438 &2821

94 % Cgkat1 2 heme catalase superfamily: large subunit heme catalase

CcochkatA2 1883 &2899

87 % Cgkat2 3 heme catalase superfamily: large subunit heme catalase

CcochkatB1 0511 86 % Cgkat3 2 heme catalase superfamily: small subunit heme catalase

CcochkatB2 0351 67 % SschkatE 2 heme catalase superfamily: small subunit heme catalase

* - With closest known phylogenetic neighbour** - Abbreviations of peroxidase & catalase gene names are explained in Additional file 3: Table S2, Additional file 5: Table S4, Additional file 6: Table S3,Additional file 7: Table S5 and Additional file 8: Table S6

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fungi [20] and particularly on the thermostability of Chae-tomiaceae [21] where C. cochliodes was not included at thattime.

Putative heme peroxidases & catalases in ChaetomiumcochliodesIntracellular hydrogen peroxide is a by-product ofvarious physiological pathways but, unique among allreactive oxygen species, it serves also as an import-ant signalling molecule in apoptosis and ageing [22].In filamentous fungi hydrogen peroxide was shownto be implicated in essential proliferation and differ-entiation processes [23]. Thus we have performedthis genomic screening for all possible ORFs codingfor a) enzymes supposed to release H2O2 duringtheir reaction and b) two main types of enzymes in-volved in the catabolism of hydrogen peroxide in anovel genome of a soil Ascomycete. With TBLASTXmethod we could identify 8 genes for various

oxidoreductases producing H2O2 (Table 2A) and upto 20 distinct genes belonging to various heme andnon-heme peroxidase superfamilies as well as to theheme catalase superfamily. Overview on all thesegenes together with their introns composition is pre-sented in Table 2B. All presented sequences are fromcontigs of the genome project deposited at GenBank underaccession LSBY00000000, BioProject PRJNA309375, Bio-Sample SAMN04432217. From Table 2 it is obvious thatgenes coding H2O2 degradation exhibit a higher diversitythan genes coding H2O2-releasing enzymes. Detected genesfor non-heme peroxidases include vanadium-containinghaloperoxidase, glutathione peroxidase as well as 1-cysteineand 2-cystein peroxiredoxins. This work focuses further ongenes coding for heme peroxidases.As was presented recently, there are at least four heme

peroxidase superfamilies and one heme catalase superfam-ily that arose independently during a convergent evolution.They differ in overall fold, active site architecture and en-zymatic activities [10]. The following sections aim to dis-cover all genes for representatives of all five superfamilieswithin the genome of C. cochliodes and to determine theirexact phylogenetic positions. Heme peroxidases are foundin all kingdoms of life and typically catalyse the one- andtwo-electron oxidation of a myriad of organic and inorganicsubstrates. In addition to the basal peroxidatic activity dis-tinct families show pronounced catalase, cyclooxygenase,chlorite dismutase or peroxygenase activities.

Table 3 List of primers for C.cochliodes peroxidase & catalase genes

B Primer description Sequence in 5′→ 3′ direction Tm [°C] PCR prodct size [bp]

hyBpox1 CcochhyBpox1Fwd CGAGAAAACAGATATTCTAGAAGCCA 60.1 116

CcochhyBpox1Crev TTCTACCGGCACCTAAATTGTT 56.5

hyBpox2 CcochhyBpox2aFWD GTTCATTTAGCAGGAGGTCAGG 60.3 119

CcochhyBpox2aREV TGTCACTGCTCGAGTTAGCATT 58.4

cyox1 CcochCyox1bFWD GCCTTCAAACTCCTCAACAAAG 58.4 117

CcochCyox1bREV GTAGCCGTCATGGAGGTTGTAT 60.3

lds CcochLDS3FWD AACTTACACCATCTCCCGTGTC 58.4 127

CcochLDS3REV GTCGTACTGAGCGTCGTTGTAA 60.3

dyPrx CcochDyprxBfwd2 AAAGGAATGTCGAACCAAAAGA 54.7 135

CcochDyprxBrev1 GCCGAGAGTAAAATCTGGAATG 58.4

htp1 CcochHtp1fwd2 ATCTTCAACCAGACCATCTTCG 58.4 114

CcochHtp1rev2 GAACGACTTGGACTCGATCTG 59.8

katA2 CcochkatA2_IFWD GAATCAACAAGACGCTTTGTGG 63.5 202

CcochkatA2_IREV TAGGTGGGTTAGCAAGTGAGAG 63.3

katB1 CcochkatB2_2REV TAAACACAAAGTCCTGGTTCCC 58.4 207

CcochkatB1_2REV TGGAAAAGGCGCCGTAGTCG 61.4

katB2 CcochkatB2_1FWD GGGGCGAGTTTGAGGTGACC 63.5 198

CcochkatB2_2REV TAAACACAAAGTCCTGGTTCCC 58.4

Table 4 comparison of three related Chaetomia genomes

Organism Reference Genomesize [bp]

Comparison withC.coch.

PredictedORFs

C. cochliodes this work 34,745,808 10.103

C. globosum [2] 34,886,900 100.41 % 11.048

C.thermophilum

[1] 28,322,800 81.51 % 7.165

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Peroxidase-catalase superfamilyThe peroxidase-catalase superfamily is currently themost abundant peroxidase superfamily in various geneand protein databases. It is comprised of three distinctfamilies (Families I, II and III formerly known as classes)and hybrid peroxidases that represent transition forms(clades) between these families. Here we present an up-dated reconstruction of the phylogeny of this largestknown heme peroxidase superfamily analysed previously[24, 25]. Our updated input included already 632complete sequences and is presented in Fig. 2. We focushere on the phylogenetic positions of all representatives(ORFs) found in Chaetomia.Family I of the peroxidase-catalase superfamily typic-

ally contains catalase-peroxidases (KatG), ascorbate per-oxidases and cytochrome c peroxidases (CcP) [24]. A

HGT-event from Bacteroidetes to filamentous Ascomy-cetes was previously reported as a peculiarity of katGgene family evolution [26]. Circular tree of the wholesuperfamily clearly demonstrates that all katG1 genesfrom the Chaetomiaceae family (cf. Additional file 3:Table S2 for abbreviations) apparently are late descen-dants of this HGT event (Fig. 2 left upper part). Withinthe upper clades we observe a basal position of thethermophilic variants from which their mesophilic coun-terparts descended. However, a question remainswhether only the coding region of katGs was transferredfrom bacteria to fungi or whether some neighbouring re-gions were also included in such a transfer? We demon-strate for the gene encoding KatG1 in C. cochliodes (i.e.CcochkatG1) that the regulatory elements located on 5′and 3′ regions embedding the ORF are clearly of

Chaetomium cochliodes .

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Fig. 1 Phylogenetic relationship among 34 Ascomycetes reconstructed from the conserved region spanning 18S-ITS1-5.8S-ITS2-28S rDNA genes.Maximum likelihood method from MEGA6 with 1000 bootstraps and MrBayes method over 200,000 generations were applied on the same DNA sequencealignment 2,474 bp long (Additional file 2: Figure S1). Bootstrap values above 50 & posterior probabilities are shown, respectively. Scale bare represents thefrequency of ML substitutions

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eukaryotic origin (Fig. 3). In the promoter region thereis (besides the GC box) a typical regulatory sequence –the “CCAAT” box involved in eukaryotic oxidative stressresponse [27]. In the 3′ untranslated region the poly-Asite for corresponding mRNA formation can be pre-dicted with a high probability. Thus, we can concludethat a prokaryotic katG was inserted in the fungal gen-ome but received a typical eukaryotic transcription regu-lation during later evolution. The main physiologicalrole of KatG in C. cochliodes is most propable dismuta-tion of metabolically-generated hydrogen peroxide tomolecular oxygen and water, similar to typical (mono-functional) catalases (see below) [24, 26]. In addition toKatG Chaetomia contain genes (ccp) encoding cyto-chrome c peroxidases (CcP, Fig. 2 – middle of the leftpart). The relationships among the fungi presented inthe CcP phylogenetic analysis suggest that this protein

has evolved vertically throughout Ascomycetes. For ccpgenes from both C. globosum and C. cochliodes a basallineage represented by C. thermophilum and M. thermo-phila is apparent in the reconstructed tree. The physio-logical role of CcP is still under discussion.Further phylogenetic reconstruction of the peroxidase-

catalase superfamily reveals that in C. cochliodes but notin C. globosum a Family II representative is present(Fig. 2 – lower part). This is very surprising for suchclosely related fungal species. However, the Family IIrepresentative from C. cochliodes has its closest neigh-bour in C. thermophilum. Family II ascomycetous genescode for hypothetical heme peroxidases with yet un-known reaction specificity but are closely related withwell investigated basidiomycetous manganese and ligninperoxidases (Fig. 2, labelled violet). The latter are in-volved in oxidative degradation of lignin-containing soil

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s.

81

Han

HyB

pox1

Ba.

Han

HyB

pox2

Ba.

99/1

.0 Ela

HyB

pox

As.

Hys

pHyB

pox1

As.

Hys

pHyB

pox3

As.

995056

Gpr

HyB

pox1

Chy

.

Gpr

HyB

pox2

Chy

.

99

Cun

iHyB

pox1

Ba.

Cun

iHyB

pox2

Ba.

9950

Apru

HyB

pox1

As.

VaaH

yBpo

x As

.

VdaH

yBpo

x2 A

s.

99 BparH

yBpo

x As.

GgrHyB

pox1

As.

60 BdoHyB

Pox1 A

s.

70 CfioHyB

pox2

As.

MagHyBpox1

As.

72LmaHyB

pox As.

PnoHyBpox1

As.

9964 GclHyBpox1 As.

MbruHyBpox1 As.

55 FoHyBpox1 As.

GmoHyBpox1 As.

99FpsHyBpox1 As.

GzHyBpox1 As.

99

99

MperHyBpox1 Ba.

CfioHyBpox1 As.

ChigHyBpox1 As.99

BdotHyBpox2 As.

HyspHyBpox2 As.

BfuHyBpox2 As.

SsclHyBpox As.99Basidiomycetous minor hyBpox clade

CcochHyBpox2 As.OppiHyBpox As.Talaromyces HyBpox1 cladeMvarHyBpox1 As.AmonHyBpox2 As.AalcHyBpox As.VdaHyBpox1 As.

92

TaspHyBpox1 As.TatHyBpox1 As.

99

TviHyBPox1 As.

99

HjHyBpox1 As.

99

EfeHyBpox As.

ManiHyBpox1 As.

99

99

TmiHyBpox As.

GgrHyBpox2 As.

MpoaHyBpox2 As.

99

MagHyBpox2 As.

98

NcHyBpox1 As.

NdisHyBpox1 As.

71

NtetHyBpox1 As.

63

SmaHyBpox1 As.

99

CtheHyBpox As.

PanHyBpox1 As.

TterHyBpox1 As.

CgH

yBpox2 As.

MthH

yBpox1 As.

9579

7683

60

66

65

5761

MvarH

yBpox2 As.

77

Trichosporon HyB

pox basal clade

86

GprA

Px-R

Chy.

Basidiom

yc. Mn&

LiPO

X Fam

.II

Dothideom

ycete PO

X2 clades

Pecam

PO

X A

s.

Neufusicoccum

PO

X2 clade

SchaP

OX

As.

Cco

chP

OX

2a As.

CtheP

OX

2a As.

99/1.0

MthP

OX

2 As.

98/1.0

Mam

yPO

X2a A

s.85

LfluPO

X2b A

s.75

CfioP

OX

2a As.

CgloP

OX

2a As.

95/1

.0C

higPO

X2a A

s.

98/1

.0

ME

spPO

X2a A

s.M

agPO

X2 A

s.

82/1

.0

FoP

OX

2a As.

Gm

oPO

X2a A

s.

99

VdaP

OX

2a As.

CfioP

OX

2b As.

CgraP

OX

2a As.

91 98

5998

8998

AP

x-re

late

d cl

ades

Chl

.

Fam

ily II

I sec

r. pe

roxi

dase

s Vi

.

Met

azoa

n A

Px

88

hybr

id A

PX-C

cP1

& AP

x cl

ades

hybr

id A

Px-C

cP2

clad

es

Hap

toph

ycae

CcP

Choan

ofla

gellid

a CcP

Glo

mer

omyc

ete

CcP1

Asper

gilli C

cP1

Chytri

diom

ycet

e CcP

1

Rhodo

phyta

& S

tramen

opile

s CcP

Chlorophyta C

cP

CsubCcP

1 Chl.

Mucoromyce

te CcP

Saccharomycete CcP

Glomeromycete & Chytridiomycete CcP

Aspergilli CcP2

Basidiomycetous CcPSsclCcP2 As.BfuCcP2 As. 99

PnoCcP1 As. 67AalcCcP1 As.CgraCcP1 As.GzCcP2 As.MagCcP2 As.NcCcP As.MthCcP As.

CtheCcP As.72/1.0

CcochCcP As.CgCcP As. 97/1.0

59/1.057/0.79

50/1.0

66/0.79

91

62 56 92

99

minor Stramenopilous KatG clades

99

Thalassiosira KatG clade

Proteobacterial minor KatG clade

PtrKatG St.

Euryarchaeal minor KatG clade

Firmicutes minor KatG clade

Bacteroidetes minor KatG clade

Actinobacterial KatG clade

Firmicutes KatG clade

Euryarchaeal KatG clade

Burkhodleria KatGs

PssKatG1 Pb.

Proteobacterial KatGs

Bacteroidetes KatGs

Ascomycetous KatG2s

Penicillium KatG clade

Aspergilli KatG1 clade

Fusarium KatG clade

Neurospora KatG clade

Blumeria KatG

clade

MagKatG

1 As.

GgrKatG

1 As.84/1.0

PanKatG1 As.

CtheKatG

1 As.

TterKatG1 As.

MthK

atG1 A

s.

CcochK

atG1 A

s.C

gKatG

1 As.

99/1.070/1.0

56/1.0

58/0.92

65/0.85

53/0.60

Cyanobacterial &

Planctom

ycetous

ancestral KatG

clades

0.50 HGT

0.51

hybrid B hemeperoxidases

catalase-peroxidases

cytochrome cperoxidases

secretory peroxidasesascorbateperoxidases

Fig. 2 Reconstructed phylogeny of the peroxidase-catalase superfamily with focus on newly sequenced Chaetomia ORFs. The complete tree from 632 fulllength sequences with 536 sites aligned is presented with collapsed branches that do not contain any Chaetomia sequences. Distinct subfamilies are la-belled in different colours. C. cochliodes sequences are labelled red. Values in nodes represent bootstrap values above 50 (from maximum likelihood ana-lysis) and posterior probabilities (from Mr. Bayes), respectively. Abbreviations of peroxidase names are listed in Additional file 3: Table S2. Abbreviations oftaxa: Pb, Proteobacteria; As, Ascomycota; Ba, Basidiomycota; Chy, Chytridiomycota; St, Stramenopiles; Chl, Chlorophyta; Vi, Viridiplantae

Zámocký et al. BMC Genomics (2016) 17:763 Page 8 of 15

debris and typically use Mn2+ or small organic moleculesas electron donors.Additional representatives from the peroxidase-catalase

superfamily in C. cochliodes include two paralogs of hybridB heme peroxidases discovered as a new gene family onlyrecently [25]. Hybrid-type B peroxidases are present solelyin fungi but are related to Family III (comprised of numer-ous plant secretory peroxidases, labelled green in Fig. 2)and also to Family II (fungal secretory peroxidases men-tioned above). The basal lineage for the first paralog(CcochHyBpox1) together with its closely related C. globo-sum counterpart appears among mesophilic Sordariomy-cetes (Fig. 2 upper part). The second variant(CcochHyBpox2) containing besides the peroxidase domainalso an additional C-terminal WSC (sugar binding) domainis not closely related with C. globosum ortholog (Fig. 2right). Thus, both these HyBpox paralogs are not the resultof a recent gene duplication but segregated rather early inthe evolution of fungal genomes. Transcription analysis(Table 5 & Additional file 4: Figure S2) reveals a slight in-duction of both hyBpox genes selectively with peroxyaceticacid in the cultivation medium. In contrast, previous results[4] reveal a constitutive mode of expression for distantly re-lated katG1 gene with hydrogen peroxide and peroxyaceticacid.

Peroxidase-cyclooxygenase superfamilyMembers of the peroxidase-cyclooxygenase superfamily(comprised of Families I - VII) are widely distributed amongall domains of life. In many cases they are multidomainproteins with one heme peroxidase domain [10, 28]. FamilyIV is comprised of bifunctional cyclooxygenases possessingboth peroxidase and cyclooxygenase activities. They are in-volved in various physiological and pathophysiological pro-cesses [29]. In mammals they are located in the luminalmembrane of the endoplasmatic reticulum and mediate theconversion of free essential fatty acids to prostanoids by atwo-step process [30]. The structure and function of thetwo distinct human paralogs (constitutive COX-1 and indu-cible COX-2) were intensively investigated but a compre-hensive analysis of their diverse paralogs among eukaryoticmicrobes or even among prokaryots was only recently re-ported [31]. Evolutionary relationships among fungal cyclo-oxygenase genes were not analysed in sufficient detail yet.

Our current reconstruction based on the phylogeny ofselected members from the whole superfamily (compris-ing 204 unique genes) is presented in Fig. 4. Genomeanalysis suggests the occurrence of two representativesof this superfamily in Chaetomia, a cyclooxygenase-likeenzyme and a linoleate diol synthase. Cyclooxygenasegenes from C. cochliodes and C. globosum share theirclosest phylogenetic neighbour (Fig. 4 upper part left) inthe genome of M. mycetomatis, a human pathogenicfungus that grows optimally at room temperature [32].No cyclooxygenase genes were found in thermophilicfungi so far. In contrast, the evolutionary reconstructionof another important subfamily of Family IV, linoleatediol synthases, reveals a very similar pattern for Chaeto-miaceae as already described for the previous superfam-ily. Corresponding part of the tree (Fig. 4 – upper partright) demonstrates that genes encoding linoleate diolsynthases (lds) from thermophilic fungi (M. thermophilaand C. thermophilum) represent basal lineages for

00

S

CcochkatG1

Fig. 3 Presentation of the promoter region for CcochkatG gene showing typical eukaryotic regulatory elements for a HGT-related bacterial gene.Sequence analysis was performed in Contig 0012 between positions 43,000 - 47,000 with FGENESH software [14], drawn to scale

Table 5 Transcription analysis of 9 selected genes for peroxidecatabolism in C. cochliodes recorded with RT-qPCR. Quantitativevalues representing relative changes of the transcription level wereobtained by comparison of the expression of a particular gene in30 min. induced vs. non induced samples. The constitutivelyexpressed ITS1 region was used as internal standard fornormalization

Changes in expression levels against non-induced control*

Analysed gene Sample with 5 mM H2O2 Sample with 5 mM PAA

CcochhyBpox1 1.5 x 3.0 x

CcochhyBpox2 0.3 x 1.7 x

Ccochcyox1 0.3 x 2.3 x

Ccochlds 0.4 x 1.8 x

Ccochdyprx 3.3 x 18.5 x

Ccochhtp1 2.7 x 2.9 x

CcochkatA2 1.1 x 0.5 x

CcochkatB1 0.4 x 1.1 x

CcochkatB2 0.6 x 1.9 x

* Changes in the expression levels compared to the control sample (with thereference value of 1.0) were calculated as relative quantities due to theformula RQ = 2 – ΔΔCq where Cq is the quantification cycle of each RT-qPCR re-action. Presented are average values of triplicates for each listed gene andeach inducer. Typical amplification plots and melting curves are presented inAdditional file 4: Figure S2

Zámocký et al. BMC Genomics (2016) 17:763 Page 9 of 15

corresponding genes in mesophilic Chaetomia. Only re-cently it was shown that fatty acid diol synthases areunique fusion proteins containing a N-terminal hemeperoxidase domain joined with a C-terminal P450-hemethiolate domain for conversion of unsaturated fatty acidsto dihydroxy-fatty acids [33]. These enzymes are an es-sential part of the psi factor sexual inducer cascade invarious fungi [34]. Their exact physiological role withinthe life cycle of Chaetomiaceae needs to be elucidated inthe future. Our first round of transcription analysis re-vealed around 2-fold induction of expression of bothcyox1 and lds genes in a medium with peroxyacetic acid(Table 5 and Additional file 4: Figure S2).

Peroxidase-chlorite dismutase superfamilyOur next screening within the C. cochliodes genome fo-cused on the presence of genes encoding dye-

decolorizing peroxidases (DyPs). These heme enzymeswere first isolated from soil basidiomycetes but were fur-ther shown to be present in a wide variety of fungi andbacteria [35]. DyPs catalyse the H2O2-mediated oxida-tion of a very broad substrate range. Originally, fungalrepresentatives were found to degrade bulky dyes. A de-tailed structure- and sequence-based comparison dem-onstrated that DyPs together with chlorite dismutasesand chlorite-dismutase like proteins (EfeB, HemQ) con-stitute the CDE superfamily [36], also designated asperoxidase-chlorite dismutase superfamily [10]. The re-constructed evolution of DyPs within this superfamily isshown in Fig. 5. In fungal genomes mainly representa-tives of the subfamilies DyP-type D and DyP-type B canbe found as paralogs. Interestingly, in the genome of C.cochliodes only a fused version of DyP-PFL is present,i.e. an N-terminal DyP peroxidase domain connected

Aal

cLD

S1

As.

Vlo

ngLD

S1

As.

94

Csu

bLD

S A

s.

100

Mag

LDS

1 A

s.G

grLD

S2

As.

Mpo

aeLD

S1

As.

100

100

77

Spb

rasL

DS

As.

59

GzL

DS

1 A

s.F

oLD

S1

As.

Gm

oLD

S1

As.

100

100

100

CgL

DS

1 A

s.

100/

1.0

Mth

LDS

As.

100/

1.0

Cth

eLD

S A

s.

81/0

.98

Pan

LDS

1 A

s.S

maL

DS

As.

NcL

DS

1 A

s.N

tetL

DS

1 A

s.

100

100

100/

0.92

100/

0.97

BfuL

DS1

As.

Sscl

LDS1

As.

100

Cim

LDS3

As.

Cpo

sLD

S3 A

s.

100

Acap

LDS3

As.

99

AflLDS3

As.

AorLD

S3 As.

100

Afum

LDS2

As.

NfLDS2 A

s.

10010

0

AteLD

S4 As.

99

AfumLD

S3 As.

NfLDS3 As.

100 AclL

DS3 As.

100AniLDS3 As.

AnLDS3 As.

AteLDS3 As.

AflLDS4 As.

AorLDS4 As.

100956798

10099

10065

94

CzmLDS1 As.

PnoLDS1 As.

100CimLDS1 As.

CposLDS1 As.

100AcapLDS As.

AnLDS1 As.

AniLDS1 As.

AteLDS1 As.

AflLDS1 As.

AorLDS1 As.100

AclLDS1As.

AfumLDS1 As.

NfLDS1 As.

10010068

651009610095

97

SsclLDS2 As.

GgrLDS1 As.

MagLDS2 As.100

96

99

CyegLDS As.AclLDS2 As.AniLDS2 As.

92

AorLDS5 As.CimLDS2 As.CposLDS2 As.

10056

100

100

100

GzLDS3 As.NcLDS2 As.GmoLDS3 As.

100

GmoLDS2 As.GzLDS2 As.

100

AalcLDS2 As.

100

AflLDS2 As.AorLDS2 As.

100

AnLDS2 As.AteLDS2 As.

100

100

100

98

86

RhdelLDS Mu.RhmicLDS Mu.

PaparLDS Mu.

MambLDS Mu.

McirLDS anMu.

100

100

82

100

89

LmacLDS As.

PnoLDS2 As.

100

SreLDS Ba.

UmLDS1 Ba.

100

SrosLDS1 Ba.

SrosLDS2 Ba.

100

MlpLDS1 Ba.

MlpLDS2 Ba.

MlpLDS3 Ba.

83

100

PcLDS2 Ba.

PplLDS2 Ba.

92JargLDS2 Ba.

100G

luxLDS2 Ba.

55

CcinLD

S2 Ba.

AbLDS2 Ba.

LbiLDS2 Ba.

9251

PinvLDS2 Ba.

SbreLDS2 Ba.

SlutLD

S2 B

a.

100100

77S

comLD

S2 B

a.

100

PindLD

S2 B

a.

100

PindLD

S1 B

a.

SbreLD

S1 B

a.

SlutLD

S1 B

a.

100

PinvLD

S1 B

a.

100

PcLD

S1 B

a.

AbLD

S1 B

a.

GluxLD

S1 B

a.

Scom

LDS

1 Ba.

73

CcinLD

S1 B

a.

LbiLDS

1 Ba.

99

JargLDS

1 Ba.

PplLD

S1A

Ba.

PplLD

S1B

Ba.

10097

77

7973

10088

87

PcL

DS

3 B

a..a

B 3S

DLgraJJarg

LDS

4 B

a.10

070

97

Cfa

PG

HS

2 D

e.

Eca

bPG

HS

2 D

e.97

Cpo

PG

HS

2 D

e.

55

BtP

GH

S2

De.

87

HsP

GH

S2

De.

100

Gga

PG

HS

2 D

e.

100

DrP

GH

S2

De.

Fhe

PG

HS

De.

76

98

DrP

GH

S1

De.

Gga

PG

HS

1 D

e.

Cfa

PG

HS

1 D

e.

Eca

bPG

HS

1 D

e.

BtP

GH

S1

De.

HsP

GH

S1

De.

5259

100 99

100

100

GfrC

yOx1

Cn.

GfrC

yOx2

Cn.

100

85

Cin

tPG

HS1

De.

Cin

tPG

HS2

De.

99

100

ACm

aPxD

o C

yb.

BmaP

xDo1

PlB

.

100

AeuC

yOx1

St.

AeuCyO

x2 S

t.

100

Cocht

hCyO

x Cy.

Mch

tCyO

x Cy.

100

Mpr

odCyO

x Cy.

60

Lirob

CyOx C

y.

96

Tolsp

CyOx C

y.

70

AcmaCyO

x Cy.

CyspCyO

x Cy.

LepspCyO

x Cy.

100 9054

CalspCyO

x Cy.

100

BrspCyOx Ap.

62

SuCyOx Aci.

92

MxCyOx1 Dp.

ScelCyOx Dp.

100

MsmCyOx1 Ac.

MfortCyOx Ac.MvulCyOx Ac.

100 100

AmmethCyOx Ac.AmvanCyOx Ac. 97StfulCyOx Ac. 59

SaceryxCyox Ac. 100FspCyOx1 Ac.

98MuflCyOx Ac.

100GospuCyox Ac.MneworlCyOx Ac.GorhiCyOx Ac.NoastCyOx Ac.

81

MabscCyOx Ac.McheloCyox Ac.

MimmunCyOx Ac. 100100

5986

100

60

FoCyOx1 As.GfujiCyOx As.

59

GmoCyOx1 As.

100

GzCyOx1 As.

99

NhaeCyOx As.

85

CgloCyOx As.

58

CorbCyOx As.

100

EpusCyOx1 As.

PficCyOx As.78

CzmCyOx As.

PfijCyOx As.100

56

RrufCyOx As.

90

AbraCyOx As.

62

BcomCyOx As.

CgCyOx1 As.

100/1.0

MamycCyOx As.

88/1.0

NcCyOx As.

NtetCyOx As.

100/1.0

68/1.0

AumelCyOx As.

AupulCyOx As.

71

AusubCyOx As.

89

AunamCyOx As.

100

BiochCyOx As.

StchlCyOx As.

100

BebasCyOx As.

76

StchaCyOx As.

76

FoCyOx2 As.

MeguCyOx As.

100

96

BdotCyOx As.

DahapCyOx As.

52

EpusCyOx2 As.

PspanCyOx As.

74

AteCyO

x1 As.83

MbruC

yOx As.

91

FpedCyox As.

HpulC

yOx As.

ExspiC

yOx A

s.

ExxenC

yOx A

s.

9966

10062

100/0.53

100/1.0

100

98

100

97

60 72

CseP

xc1 Prb.

FspPxc1 A

cb.100

88

AhspP

xc1 Prb.

BjaP

xc1 Prb.

88

cyclooxygenases lionoleate diolsynthases

peroxicins

pero

xido

cker

ins

Fig. 4 Reconstructed phylogeny of the peroxidase-cyclooxygenase superfamily with focus on Chaetomia ORFs. The complete tree from 204 fulllength sequences with 1,053 aligned sites is presented. C. cochliodes sequences are labelled red. Distinct subfamilies are labelled in different col-ours. Values in nodes represent bootstrap values above 50 (from maximum likelihood analysis) and posterior probabilities (from Mr. Bayes), re-spectively. Abbreviations of peroxidase names are listed in Additional file 5: Table S3. Abbreviations of taxa: Ac, Actinobacteria; Acb, Acidobacteria;Cy, Cyanobacteria; Prb, Proteobacteria; Plb, Planctomycetes (bacteria); As, Ascomycota; Ba, Basidiomycota; Mu, Mucoromycota; St, Stramenopiles;Cn, Cnidaria; De, Deuterostomia

Zámocký et al. BMC Genomics (2016) 17:763 Page 10 of 15

with a C-terminal pyruvate formate-lyase (PFL) domainknown as a glycyl radical containing region [37]. Thisunique gene fusion was detected also in other distantlyrelated prokaryotic & eukaryotic genomes [38]. The PFLdomain can be activated by PFL activase, a radical SAMsuperfamily member [39], but the significance of a PFLfusion with a peroxidase domain remains elusive. Wecould detect a putative PFL activase in C. cochliodescontig 00230 revealing 81 % identity with CHGG_03160from C. globosum and other putative PFL activases fromfilamentous fungi. Thus, C. cochliodes possesses both

components necessary for the glycyl radical formationwith yet unknown physiological function. A HGT eventwith a high bootstrap support in the clade of fused DyPsB can be observed between proteobacteria and ascomy-cetous fungi (Fig. 5 and Additional file 6: Table S4 forabbreviations). As the fused DyP B-PFL proteins are yethypothetical, their physiological relevance has to be de-termined among Chaetomiaceae. Our first round oftranscription analysis of dyprx gene exhibited the highestinduction observed among all 5 superfamilies followedin this study with hydrogen peroxide (3-fold) and mainly

BaD

yPrx

3 B

a.

TcuD

yPrx

1 B

a.

99

Pbr

eDyP

rx3

Ba.

72

Pol

ypP

rx B

a.Ta

lDyP

rx B

a.99

99

PoD

yPrx

1 B

a.P

inD

yPrx

As.

Ade

lDyP

rx7

Ba.

Aau

rDyP

rx B

a.E

xgla

DyP

rx B

a.

74

9960

Mpe

rDyP

rx2

Ba.

Msc

DyP

rx1

Ba.

Glu

xDyP

rx7

Ba.

77

98

92

Cci

nDyP

rx1

Ba.

Am

usD

yPrx

1 B

a.

75

Glu

cDyp

Prx1

Ba.

Gsp

pDyP

rx2

Ba.

99

97

53

Mlp

Dyp

rx1

Ba.

91

AflD

yPrx

As.

AorD

yPrx

As.

99

Afum

DyPrx

As.

NfDyP

rx A

s.

99

99

Pechr

DyPrx

2 As.

AclDyP

rx2 A

s.

CzmDyP

rx As.

Byssp

DyPrx

As.

SsclD

yPrx

As.

SborDyP

rx As.

BfuDyPrx

As.

91 NcDyPrx As.

SmaDyPrx As.

SmaDyPrx2 As.

9999969799996896

73

LbiDyprx2 Ba.

PplDyPrx1 Ba.

99

99

NospuDyprx Cy.

AcmaDyPrx Cy.

RbaDyPrx Pmc.

8598AcspDyPrx Ac.

AcmiDyPrx Ac.99

MeteyDyPrx Pb.

RhlegDyPrx Pb.86

SlingDyPrx Bi.98

AvarDyPrx Cy.

RbrooDyPrx Cy.

CyanspDyPrx2 Cy.

CyanspDyprx3 Cy.67

99

99

CycmarDyPrx Bi.ChDyprx Bi.

52

NeDyPrx Pb.NimulDyPrx Pb.

99

57

MoprDyPrx Cy.MealFusDyPrx Pb.

90

CyanspDyPrx1 Cy.VniDyPrx Pb.VibnigDyPrx Pb.

99

AcspDyPrx Aci.

99

PpacDyPrx Pb.

58

OantDyPrx Pb.MopeDyPrx Ac.

67

MiluDyPrx Ac.

99

AmymedDyPrx Ac.

DraDyPrx Dei.

88

NocaDyPrx Ac.

SsvDyPrx Ac.

99

81

CgleDyPrx Bi.

PsspDyPrx Pb.

AclwDyPrx Pb.

EnaeDyPrx Pb.

99

99

99

92

KoverDyPrx Aci.

CalspDyPrx Cy.

96M

smeDyPrx Ac.

MYspDyPrx3 Ac.

9999

SavDyprx3 Ac.

KteracDyprx C

hl.

FrspDyPrx Ac.

SusDyPrx Aci.

68

SocD

yPrx P

b.

58C

yfusDyP

rx Pb.

MxD

yPrx P

b.

CorcorD

yPrx P

b.

9291

9962

99

93

98

99

PaninD

yPrx vir.

PandulD

yPrx vir.

57

PansalD

yPrx vir.

99

AccasD

yPrx A

mb.

99

CtesD

yPrx1 P

b.

PputD

yPrx P

b.

PfD

yPrx2 P

b.78

99

Sch

japD

yPrx

Tre

.S

chm

anD

yPrx

Tre

.99

.l oM xr

PyDl uvr

A

99

Ngr

DyP

rx1

Am

b.

Pte

tDyP

rx A

lv.

81

63

Ngr

DyP

rx2

Am

b.

54

BfD

yPrx

1 D

eu.

SflD

yPrx

2 P

b.

EcD

yPrx

2 P

b.

99

Gal

anaD

yPrx

Pb.

97

DdD

yPrx

Myc

.

87

Vib

lenD

yPrx

Pb.

Vib

chol

DyP

rx P

b.99

Son

DyP

rx P

b.

56

Acs

pDyP

rx P

b.

Bth

eDyP

rx B

i.

Pdi

DyP

rx B

i.

67

BfrD

yPrx

Bi.

99

Cco

nDyP

rx P

b.

Ccr

vDyP

rx P

b.99

89

Cta

iDyP

rx P

b.

ZmoD

yPrx

Pb.

Rhosp

DyPrx

2 Ac

.

RjoDyP

rx A

c.

99

TasD

yPrx

Ba.

Mlae

DyPrx

Ac.

GzDyP

rx A

s.

PmDyPrx

As.

FoDyPrx

As.

GmoDyPrx1

As.99

NhaeDyPrx

As.98

CgloDyPrx

As.

89

ManiDyPrx As.

MacrDyPrx As.

99

ClpsDyPrx As.

CacorDyPrx As. 94

TminDyPrx As.AflDyPrx1 As.

AorDyPrx1 As. 99ByspecDyprx As.OmaiDyPrx As.HpulDyPrx As.PEchDyPrx1 As.

7599 59 99 60

99

63

EcEfeB Pb.SboDyPrx1 Pb.

99

CityoPrx Pb.82

KpnDyPrx Pb.

99

YpDyPrx1 Pb.

99

OantDyPrx2 Pb.

RpDyPrx Pb. 95

99

CaacDyPrx Ac.

SthygDyprx Ac. 99

94

MsmDyPrx1 Ac.

MvaDyPrx1 Ac.

74

NfaDyPrx1 Ac.

99

TfuDyPrx Ac.

TcelDyPrx Ac.99

82

GesaDyPrx Fi.

LhofDyPrx Fb.

SsaDyPrx Fi.

StheCldL Fi.99

95

AbdeDyPrx Fi.

99

BsDyPrx Fi.

79

PcurDyPrx Fi.

PaespDyPrx Fi.

99

BxDyPrx1 Pb.

GobronDyPrx Ac.

RhospDyPrx1 Ac.

89

MispDyPrx Ac.

ThbiDyPrx Ac.

99

99

88

90

9771

RsoDyPrx Pb.

AcicalcDyPrx Pb.

CviolDyPrx Pb.

CocorDyPrx Pb.

9399

55

CollareDyPrx Pb.

JaagaDyPrx Pb.

95

DespD

yPrx Pb.

CcochD

yPrx As.

CgD

yPrx As.99/1.0

GfujiD

yPrx A

s.

Gm

oDyP

rx2 As.

99/1.094/1.099/1.0

99H

aantDyP

rx Pb.

BfD

yPrx2 D

eu.

98

clades of Cld &

CldL

(bacterial & archaeal)

fusions DyP-PFL

dye-decolorizingperoxidases (DyPs)

HG

T

Fig. 5 Reconstructed phylogeny of the peroxidase-dismutase superfamily with focus on newly discovered Chaetomia sequences forming a separate cladeof DyP-Bs together with fused bacterial representatives from which they were derived by a HGT event. The complete tree from 282 full length sequencesis presented with 655 sites aligned. C. cochliodes sequence is labelled red. Distinct subfamilies are labelled in different colours. Values in nodes representbootstrap values above 50 (from maximum likelihood analysis) and posterior probabilities (from Mr. Bayes), respectively. Abbreviations of peroxidase namesare listed in Additional file 6: Table S4. Abbreviations of taxa: vir, DNA viruses; Ac, Actinobacteria; Aci, Acidobacteria; Bi, Bacteroidetes; Chl, Chloroflexi (bac-teria); Cy, Cyanobacteria; Dei, Deinococci; Fi, Firmicutes; Pb, Proteobacteria; Pmc, Planctomycetes; As, Ascomycota; Ba, Basidiomycota; Alv, Alveolata; Amb,Ameboflagellates; De, Deuterostomia; Mol, Mollusca

Zámocký et al. BMC Genomics (2016) 17:763 Page 11 of 15

with peroxyacetic acid (18.5-fold) in the cultivationmedium (Table 5).

Peroxidase-peroxygenase superfamilyHeme-thiolate peroxidases from Fungi and Stramenopilesconstitute the peroxidase-peroxygenase superfamily [10].Enzymes encoded by htp genes represent probably themost versatile catalysts among peroxidase superfamiliesthus catalysing on one side classical heme peroxidase reac-tions and on the other side monooxygenase (monohydroxy-lation) reactions like cytochrome P450s [40]. Thereconstructed phylogenetic tree for the peroxidase-peroxygenase superfamily (Fig. 6) reveals the distribution ofthree gene paralogs of this superfamily within the Chaeto-mium cochliodes genome. The presence of multiple geneparalogs in genomes of ascomycetous fungi is frequent andoccurred by repeated gene duplications of this rather shortgene but the phylogenetic distribution of C. cochliodes

paralogs is variable (Fig. 6). Whereas there is a thermophilicbasal lineage for CcochHTP2 and CcochHTP3 and theircorresponding counterparts in C. globosum, the situationfor paralog CcochHTP1 is different. Corresponding genesfrom pathogenic fungi represent a basal lineage for closelyrelated CcochHTP1 and CgHTP1. It is unknown so farwhether these three putative heme-thiolate peroxidases ex-hibit different enzymatic properties but they were segre-gated early during the evolution of fungal genomes andthus they all may be interesting for biotechnological appli-cations. We have also performed transcription analysis ofhtp1 gene paralog resulting in almost 3-fold induction bothwith hydrogen peroxide and peroxyacetic acid present inthe cultivation medium (Table 5).

Putative heme catalases in ChaetomiaTypical (monofunctional) heme catalases are enzymesthat very efficiently dismutase hydrogen peroxide to

AnH

TP

1 A

s.

2P

THcif

PA

s.O

mai

HT

PA

s.T

terH

TP

1 A

s.67

Stc

harH

TP

As.

Stc

hlH

TP

As.

100

CgH

TP

2 A

s.C

coch

HTP

2 A

s.C

gHT

P3

As.

100/

1.0

Mth

HT

PA

s.Th

hetH

TP1

As.

100/

1.0

72/0

.55

100/

1.0

Ela

HTP

As.

71

Aor

HTP

1 A

s.P

braH

TP1

As.

53

92

Pno

HTP

2 A

s.

68

Dse

pHTP

1 As

.

Spm

usH

TPAs

.

83

AniH

TP3

As.

Bcin

HTP

1 As

.

SsclH

TP1

As.

100

Ccoch

HTP1

As.

CgHTP

1 As.

99/1

.0 Mam

yHTP1 A

s.

89/1

.0

Mpo

aeHTP

As.

100/

1.0

PnoHTP3 A

s.

AniHTP2 As.

AteHTP2 As.

AorHTP2 As.

AparaHTP As.

10086

9660

93/0

.8

97

75

79/0

.54

PnoHTP1 As.

AniHTP1 As.

PficHTP1 As.

55AteHTP1 As.

GzHTP1 As.

6750100

65ColfioHTP As.

ChigHTP2 As.

96

ColgloeoHTP As.

ChigHTP1 As.

6610077

GfujHTP1 As.

GmoHTP1 As.100

GzHTP2 As.88

PangHTP4 As.75

NcHTP1 As.79

MagHTP1 As.100

AniHTP5 As.

CcochHTP3 As.CgHTP4 As.

100/1.0

TislHTP1 As.TterHTP2 As.

100/0.97

100/1.0

60/0.89

MroUPO1 Ba.MrorHTP4 Ba.

91

GluxHTP1 Ba.GluxHTP3 Ba.

64

GluxHTP2 Ba.

100

CytorHTP1 Ba.MrorHTP1 Ba.MrorHTP5 Ba.

MrorHTP6 Ba.

94

MperHTP1 Ba.MrorHTP2 Ba.

MrorHTP3 Ba.

62

73

100

46

73

100

77/0.79

PnoHTP4 As.

AniHTP4a As.

AniHTP4b As.

100

PnoHTP5 As.

HminHTP1 As.

OpsiHTP1 As.

OpsiHTP2 As.

76

100

CfumagHTP1 As.

CfumagHTP2 As.

100HjHTP1 As.

TharHTP1 As.

100AphasHTP St.

AphinHTP2 St.

100SdicH

TP1 St.

82AphinH

TP1 St.

98A

kawH

TP1 A

s.

AorH

TP3 A

s.

Afum

HTP

1 As.

91P

oxalHTP

1 As.

100G

zHTP

3 As.

NhaeH

TPA

s.

100100

MuccirH

TP

Mu.

RdelH

TP

1 Mu.

RorH

TP

1 Mu.

100100

67

AbH

TP

2 B

a.Lb

iHT

P4

Ba.

59

Cci

nHT

P3

Ba.

100

Pin

vHT

P3

Ba.

Ppl

HT

P4

Ba.. a

B3

PT

HcP

.aB

4P

THrac

P

PcH

TP

2 B

a.78

97

90

98

Ppl

HT

P5

Ba.

82

LbiH

TP

3 B

a.

Pin

vHT

P4

Ba.

Slu

tHT

P4

Ba.

100

92

94

SA

coH

TP

1 A

s.

SA

coH

TP

2 A

s.10

0

Pra

mH

TP

1 S

t.

Ppa

rHTP

1 S

t.

Pso

jHTP

1 S

t.

100

Pul

HTP

1 S

t.

51

Pso

jHTP

2 S

t.

Pul

HTP

2 S

t.

PiH

TP2

St.

Ppar

HTP

3 St

.

100

Psoj

HTP

3 St

.

Pram

HTP

2 St

.

PiHTP

1 St

.

PparH

TP2

St.

98

100 10

0

70

69

93

RirHTP

1 M

u.

PhubH

TP1

Ba.

UmHTP3

Ba.

80

SreHTP1 B

a.

98

PsanHTP1 B

a.

UhHTP1 Ba.

100

MlarHTP1 Ba.

MellpHTP Ba.

100 99

DacspHTP1 Ba.

CsubHTP1 Ba.

PnoHTP7 As.PplHTP1 Ba.

PplHTP3 Ba. 100

PplHTP2 Ba. 100

PcarHTP1 Ba.PcHTP1 Ba.100

HirHTP1 Ba.DsquaHTP1 Ba.CcinHTP1 Ba.CcinHTP2 Ba.100

AbHTP1 Ba.LbiHTP1 Ba.LbiHTP2 Ba. 100

PinvHTP1 Ba.PinvHTP2 Ba.

98

SerlaHTP1 Ba.

SbreHTP2 Ba.

SlutHTP2 Ba.

100

SbreHTP1 Ba.

SlutHTP1 Ba.

100

SbreHTP5 Ba.

SlutHTP5 Ba.99

72

54

67

56

60

89

9988

AfumHTP3 As.

AorHTP4 As.

100

AfumHTP2 As.

AteHTP3 As.

100

PnoHTP13 As.

99

GzHTP4 As.

GzHTP5 As.

PnoHTP9 As.

MpiHTP1 As.

PnoHTP10 As.

77

PnoHTP12 As.

PanHTP1 As.

PnoHTP8 As.

PnoHTP11 As.

93

100

65

PnoHTP6 As.

SreHTP2 Ba.

UmHTP1 Ba.

78

UmHTP2 Ba.

100

AdelHTP1 Ba.

AdelHTP5 Ba.100

CopradAPO

1 Ba.

CcinH

TP4 Ba.

AbHTP5 Ba.

AaeAPO1 2yorBa.

AaeA

PO

2 Ba.

96

AbH

TP3 B

a.A

bHTP

4 Ba.

100

Gm

argHTP

1 Ba.

LbiHTP

5 Ba.

5671

100

86

peroxidase-peroxygenases (HTPs)

- basidiomycetous

- ascomycetous

- mucoromycetous- oomycetous

- ascomycetous

- basidiomycetous

- ascomycetous

- basidiomycetous

Fig. 6 Phylogeny of the peroxidase-peroxygenase superfamily representing numerous gene paralogs of this superfamily among Chaetomiaceae.The complete tree from 172 full length sequences is presented with 287 sites aligned. C. cochliodes paralogs are labelled red. Distinct subfamiliesare labelled in different colours. Values in nodes represent bootstrap values above 50 (from maximum likelihood analysis) and posterior probabil-ities (from Mr. Bayes), respectively. Abbreviations of peroxidase names are listed in Additional file 7: Table S5. Abbreviations of taxa: As, Ascomy-cota; Ba, Basidiomycota; Mu, Mucoromycota; St, Stramenopiles

Zámocký et al. BMC Genomics (2016) 17:763 Page 12 of 15

oxygen and water. In contrast with heme peroxidasesthey can both reduce and oxidize hydrogen peroxide andhave negligible peroxidatic activity [41]. Heme catalasesrepresent a monophyletic group that evolved as a dis-tinct gene family from prokaryotes to almost all lineagesof eukaryotes [11]. In Fig. 7 the phylogeny focused onfungal heme catalases is presented. There are 3 distinctclades of genes for typical catalases defined by Klotz etal. [42]. In fungi only representatives of Clade 2 (largesubunit, secretory catalases) and Clade 3 (small subunit,mostly peroxisomal catalases) can be found. There areup to four gene paralogs of a catalase gene within C.cochliodes genome that underlines the importance ofmostly monofunctional catalases for the removal ofH2O2. There are thermophilic basal lineages for the largesubunit secretory catalases CcochKatA1, CcochKatA2

and their C. globosum counterparts, a situation verysimilar to the peroxidase superfamilies. In contrast,there are mesophilic basal lineages for the small sub-unit peroxisomal catalases CcochKatB1 and Ccoch-KatB2 (Fig. 7 – on the right). In particular,CcochKatB1 and CgKatB1 have a basal lineage amongcatalases from various soil and phytopathogenic fungi.Surprisingly, CcochKatB2 has no counterpart in theclosely related genome of C. globosum. Putative cata-lase from a widely distributed soil fungus S. schenckiishares a common ancestor with this unique smallsubunit peroxisomal catalase of C. cochliodes (Fig. 7).Possible involvement of C. cochliodes four catalaseisozymes in the defence against oxidative stress wasanalysed by RT-PCR. Obtained results in the early ex-ponential phase of fungal growth show only a slight

HsK

atA

Dt.

.tD

1taKort

P94

Pab

eKat

1 D

t.

99

Caj

aKat

1 D

t.

99

BtK

atA

Dt.

Cfa

Kat

1 D

t.S

scK

at1

Dt.

Mm

Kat

1 D

t.

100

XlK

at2

Dt.

XtK

at1

Dt.

XlK

at1

Dt.

100

66

70

Km

aKat

1 D

t.D

rKat

1 D

t.H

mol

Kat

1 D

t.

100

99

100

Dm

aKat

1 E

c.P

vanK

at1

Ec.

Avi

Kat

1 C

nTa

dKat

1 P

l.

69

Aae

Kat

1 E

c.

AaeK

at2

Ec.

100

AgaK

at1

Ec.

100

Bmor

Kat1

Ec.

DpK

at1

Ec.

Dm

KatE

1 Ec

.

100

80

100

HcoKa

t1 E

c.

CelKat

1 Ec.

CbrKat

1 Ec.

100

100

100

TgKat

1 Ap.

74

RoKat

1 Zy.

PblKat

1 Zy.

100 Ngr

Kat1 H

l.

PtetKat1

Ci.

TthKat1 Ci.

100 CowKat1 Ich.

PpalKat1 My.

DdKat1 My.

DpuKat1 My.

56100

4472

MarKat1 Ar.

MbaKat1 Ar.

MbuKat1 Ar.

10084

BsKat1a Fi.

BsKat1b Fi.

100KraKat1 Chl.

86 MpaKat1 Bi.

PpuKat1 Pb.

PpuKat2 Pb.

98

PfKat1 Pb.

100SgKat1 Pb.

100NOpuKatE Cy.

RmaKat Bi.

CjejKat1 Pb.

SmeKat1 Pb.46

HaurKat1 Chl.80

PproKatE Pb.

VpKat1 Pb.

PaerKat1 Pb.100

7060

98

SmKat3a Cryp.

PpaKat5 Cryp.PpaKat3 Cryp.PpaKat4 Cryp.74

100100

LplKat1 Fi.SerKat1 Ac.CgluKatE Ac.CjeKatE Ac.

10099

86

81

CgaKat1 Ba.CnKat4 Ba.

100/1.0

ArolKat1 As.CcochKatB2 As.

100/1.0

SschKat1 As.

67/0.93

NcKat4 As.GzKat2 As.GmoKat1 As.

100

65

74

PechKatE1 As.

100

TorhemKat1 As.

ScboKat1 As.HarKat1 As.

CcochKatB1 As.

CgKatB1 As.

100/1.0

100/1.0

53/1.0

100/1.0

80

DhKat1 As.PguKat1 As.

100

SceKatT As.

KlKat1 As.

100

81

YlKat1 As.

99

PgrKat1 Ba.

GzKat4 As.

98

AniKat2 As.

PEchKatC As.

100

CgloKat1 As.

100

SpomKat1 As.

50

CboiKatAAs.

AgoKat1 As.

SceKatAAs.

100CalKat1 As.

PstiKat1 As.

10055

9760

75

97

91

PcKat3 Ba.

CcinKat4 Ba.

CtherKat3 As.

CheKat1 As.

PnoKat1 As.

100

100100

100

92

BhaK

at2 Fi.

BsK

at2b Fi.

100B

deKat1 C

hy.

100P

fKat2 P

b.

81Zm

Kat3 M

ctd.

SbK

at1 Mctd.

100

OsK

at1 Mctd.

100A

tKat3 E

udi.

CreK

at1 Chlph.

PpaK

at1 Cryp.

PpaK

at2 Cryp.

100

Zm

Kat2 M

ctd.

Sm

Kat1a C

ryp.

Sm

Kat01b C

ryp.100

AcvK

at2 Cryp.

83

MpK

at1 Cryp.

82

AtrK

at1 BM

agno..ong

aM

B2t

aKr

tA

50

OsK

at2 Eudi.

Zm

Kat1 M

ctd.

100

ZaK

at1 Mctd.

AtK

at1 Eudi.

NtK

at1 Eudi.

AtK

at02 Eudi.

AlyK

at2 Eudi.

100

PdelK

at2 Eudi.

PpeK

at1 Eudi.

GhK

at1 Eudc.

PtK

at1 Eudi.

5199

61

92

6451

5351

5995

10091

77

Cco

chK

atA

2 A

s.

CgK

atA

2 A

s.

99/1

.0

Mth

Kat

2 A

s.

Thhe

tKat

1 A

s.10

0/1.

0

97/1

.0

Mam

yKat

2 A

s.

62/0

.72

Sct

heK

at1

As.

Cth

erK

at2

As.

Pan

Kat

B A

s.

88/0

.72

NcK

at3

As.

Cgr

aKat

2 As

.

GzK

at3

As.

Cpu

rKat

2 As

.

90

67

58

Mag

KatE

2 As

.

PnoK

at3

As.

AnKat

B As.

PEchKat

3 As.

100

100

RoKat

2 M

u.

PblKat

2 Zy.

100

89

SmeK

at2

Pb.

Ccoch

KatA1 A

s.

CgKatA

1 As.

100/1

.0

MthKat1 As.

TterKat1 As.

1.053/0.66

MamyKat1 As.

PanKatAAs.

50/1.0

NcKat1 As.

CtherKat1 As.

100

CfulKat2 As.

MagKatE1 As.GzKat1 As.

NhaeKatE1 As. 93

CgraKat1 As.PnoKat2 As.

CheKat2 As.PtritK

at1 As.99 100

61BfuKat1 As.SsclKat1 As.100

89AcapKat1 As.CimKat1 As. 91AniKat1 As.AnKat1 As.AfumKat1 As.AorKat1 As.

6097

100

100

PoKat2 Ba.100

CnKat1 Ba.100

DdKat2 My.DpuKat2 My.

100

EcoKatE Pb.SetyKatE Pb.

100

LAinKat1 Pb.

AkmKat1 Ver. 100

87

SscaKat1 Ac.

SsvKat1 Ac.100

STroKat1 Ac.

SerKat2 Ac. 53

MavKat1 Ac.

NfaKatB Ac.100

FspKat1 Ac.

100

DalKat1 Fi.

BhaKat1 Fi.

97

BsKat2a Fi.

BalKat1 Fi.100

57

MmazKat1 Ar.

MEmaKat1 Ar.100

90

OiKat Fi.

BdeKat2 Chy.

100

EXsKat1 Fi.

BanKat1 Fi.

BceKat1 Fi.100

100

63

97

PpuKat3 Pb.

PfKat3 Pb.

100

BphyKat1 Pb.

BpKat1 Pb.

100

99

VpaKat1 Pb.

POsKat1 Pb.

100

CatKat1 Bi.

FbKat1 Bi.

CHglKat1 Bi.

SpliKat1 Bi.

PEheKat1 Bi.

FjKat1 Bi.

CHpiKat1 Bi.

PEspKat1 Bi.

DYfeKat1 Bi.

9862

81

59

52

67

79

95

100

CvirKatLox1 C

n.

GfrKatLox C

n.93

PhoKatLox1 Cn.

100

NO

spKatLox1 Cy.

AC

maK

atLox1 Cy.

Sm

KatFus C

ryp.

SbiK

atFus Mctd.

OsjapFus M

ctd.100

RcFus E

udi.V

vKatFus E

udi.98

100100

6395

100

1.0

catalases

-Clade 3 small subunit, peroxisomal

-Clade 1 small subunit

-Clade 2 large subunit, secretory

-catalase-lipoxygenasefusion

Fig. 7 Reconstructed phylogeny of the heme catalase super family with focus on Clade 2 and 3 representing the distribution of Ascomycetouslarge subunit as well as small subunit catalases (labelled in different colors). The complete tree from 222 full length sequences is presented with546 sites aligned. C. cochliodes paralogs are labelled red. Distinct clades are labelled in different colours. Values in nodes represent bootstrapvalues above 50 (from maximum likelihood analysis) and posterior probabilities (from Mr. Bayes), respectively. Abbreviations of peroxidase namesare listed in Additional file 8: Table S6. Abbreviations of taxa: Ar, Archaea; Ac, Actinobacteria; Aci, Acidobacteria; Bi, Bacteroidetes; Chl, Chloroflexi(bacteria); Cy, Cyanobacteria; Dei, Deinococci; Fi, Firmicutes; Pb, Proteobacteria; Pmc, Planctomycetes; As, Ascomycota; Ba, Basidiomycota; Chy,Chytridiomycota; Zy, Zygomycota; Cn, Cnidaria; Ich, Ichthyosporea; Chlph, Chlorophyta; BMagno, basal Magnoliophyta; My, Mycetozoa; Cryp,Cryptogams, Eudi, Eudicotyledons, Mctd, Monocotyledons; De, Deuterostomia; Ec, Ecdysozoa

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induction of the paralog katB2 in the medium con-taining peroxyacetic acid (Table 5).

ConclusionsIn conlusion genomic sequence analysis revealed thatChaetomium cochliodes is closely related to C. globosum &C. elatum. These three filamentous fungi are mesophilicbut probably have thermophilic ancestors as revealed fromtheir basal lineage. C. cochliodes contains heme peroxidasesand catalases from all so far described superfamilies. Asco-mycetous genes encoding catalase-peroxidase and dye de-colorizing peroxidase were obtained during the evolutionby horizontal gene transfer from various bacteria. Severalheme peroxidases of Chaetomia like hybrid heme B perox-idase, linoleate diol synthase or DyP-type B form fusionswith additional functional domains that might enable abroader catalytic variability. Furthermore cytochrome c per-oxidase, manganese and three paralogs of heme-thiolateperoxidases are found in addition to typical (monofunc-tional) catalases of large and small subunit architecture.Our transcription analysis reveals the highest induction of afused dyprx gene with hydrogen peroxide and mainly withperoxyacetic acid in the cultivation medium followed bymoderate inductions of htp1 and hyBpox1 genes.

Additional files

Additional file 1: Table S1. Substitution models with the lowestBayesian information criterion scores for all 5 superfamilies analysed inthis contribution. (XLSX 70 kb)

Additional file 2: Figure S1. DNA sequence alignment of genomic DNAfrom 34 ascomycetous fungi in the region covering 18S rDNA – ITS1 – 5.8SrDNA – ITS2 – 28S rDNA (fasta format). (FAS 85 kb)

Additional file 3: Table S2. Abbreviations of peroxidase gene namesused for the peroxidase-catalase superfamily. (XLSX 54 kb)

Additional file 4: Figure S2. A typical profile of real-time quantitativePCR analysis of transcripts from peroxidase genes obtained from C.cochliodes under oxidative stress. Upper panel: amplification plots forhyBpox1, cyox1 and lds genes detected with SYBR Green Master Mix (AgilentTechnologies). Lower panel: melting curves for hyBpox1, cyox1 and lds genespresented in Table 5. (TIF 450 kb)

Additional file 5: Table S3. Abbreviations of peroxidase gene namesused for the peroxidase-cyclooxygenase superfamily. (XLSX 27 kb)

Additional file 6: Table S4. Abbreviations of peroxidase gene namesused for the peroxidase-dismutase superfamily. (XLSX 23 kb)

Additional file 7: Table S5. Abbreviations of peroxidase gene namesused for the peroxidase-peroxygenase superfamily. (XLSX 18 kb)

Additional file 8: Table S6. Abbreviations of catalase gene names usedfort he catalase superfamily. (XLSX 22 kb)

AbbreviationsCcP: Cytochrome c peroxidase; CldL: Chlorite dismutase-like protein;CTAB: Hexadecyltrimethylammonium bromide; HGT: Horizontal gene transfer;HMM: Hidden Markov model; KatG: Bifunctional catalase-peroxidase;LDS: Linoleate diol synthase; LiPOX: Lignin peroxidase; ML: Maximumlikelihood phylogeny; MnPOX: Manganese peroxidase; ORF: Open readingframe; PAA: Peroxyacetic acid; PEG: Polyethylene glycol; PFL: Pyruvateformate-lyase; RT-qPCR: Quantitative real-time PCR; SOD: Superoxidedismutase; WSC: Cell-wall integrity & stress response component

AcknowledgementsOur research was supported by the Austrian Science Fund (FWF, projectP27474-B22), by the Slovak Grant Agency VEGA (grant 2/0021/14) and by theSlovak Research and Development Agency (grant APVV-14-0375). We thankthe company Hermes LabSystems for providing us with AriaMx6 device.

Availability of data and materialsAll used DNA sequences are deposited in GenBank (Table 1). All proteinsequences that were used for reconstruction of phylogenies are listed inAdditional file 3: Table S2, Additional file 6: Table S4, Additional file 5: TableS3, Additional file 7: Table S5 and Additional file 8: Table S6. If possible theirPeroxiBase accession number is given to find them at (http://peroxibase.toulouse.inra.fr) if no PeroxiBase accession numbers exist yet theirUniProt (http://www.uniprot.org) accession numbers are given.

Authors’ contributionsMZ selected the fungus, designed all experiments, performed all molecularphylogeny analyses and wrote the manuscript; AK cultivated the fungus andperformed genomic & transcription analyses; KC optimised the isolation offungal DNA and performed genomic & transcription analysis; KL preparedthe genomic DNA for sequencing and contributed to the discussion; HTperformed the sequencing and assembled the contigs; CO evaluated theclassification and phylogeny of peroxidases & catalases and finalized themanuscript. All authors read and approved the final manuscript.

Competing interestsThe authors’ declare that they have no competing interests.

Consent to publishNot applicable (this manuscript does not contain any individual personsdata).

Ethics approval and consent to participateNot applicable for this fungal genomic study. None of here analysed genesof Chaetomia was used in experimental cloning research (yet).

Author details1Department of Chemistry, Division of Biochemistry, University of NaturalResources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. 2Instituteof Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21,SK-84551 Bratislava, Slovakia. 3Department of Biotechnology, University ofNatural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.

Received: 16 August 2016 Accepted: 22 September 2016

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