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

RESEARCH ARTICLE Open Access Genome sequence of the filamentous soil cochliodes reveals abundance of genes for from all and superfamilies Marcel Zámocký1,2*, Hakim Tafer3, Katarína Chovanová2, Ksenija Lopandic3, Anna Kamlárová2 and Christian Obinger1

Abstract Background: The ascomycetous family (class ) includes numerous soilborn, saprophytic, endophytic and pathogenic fungi which can adapt to various growth conditions and living niches by providing 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 contigs with an average size of 5756 bp and reconstructed its phylogeny. We show that this filamentous fungus is closely related but not identical to and . We screened and critically analysed this genome for open reading frames coding for essential antioxidant enzymes. It is demonstrated that the genome of C. cochliodes contains genes encoding putative enzymes from all four known heme peroxidase superfamilies including bifunctional catalase-peroxidase (KatG), (CcP), manganese peroxidase, two paralogs of hybrid B (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 be introduced into ascomycetes genomes by horizontal gene transfer from various . In addition, two putative large subunit secretory and two small-subunit typical are found in C. cochliodes. We support our genomic findings with quantitative transcription analysis of nine peroxidase & catalase genes. Conclusions: We delineate molecular phylogeny of five distinct gene superfamilies coding for essential heme in Chaetomia and from the transcription analysis the role of this antioxidant enzymatic armory for the survival of a peculiar soil ascomycete in various harsh environments. Keywords: Chaetomium cochliodes, Peroxidase-catalase superfamily, Peroxidase-cyclooxygenase superfamily, Peroxidase- superfamily, Peroxidase-peroxygenase superfamily, Heme-catalase super family

* Correspondence: [email protected] 1Department of Chemistry, Division of Biochemistry, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria 2Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia Full 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 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the 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 Page 2 of 15

Background University, Faculty of Natural Sciences in Brno, Czech The ascomycetous family of Chaetomiaceae (class Sor- Republic. The composition of the incubation medium dariomycetes) includes numerous soilborn, saprotrophic, and the growth conditions were the same as described endophytic and pathogenic fungi that apparently exhibit previously [4]. a large flexibility in their adaptation to various growth Genomic DNA from 100 mg of frozen fungal myce- conditions and living niches. In Mycobank (www.myco- lium was isolated with the method of Carlson [12] by bank.org) currently up to 451 members of this abundant using 2 % CTAB in a modification suitable for genome fungal family are registered but only from two represen- sequencing described in [13]. Finally, extracted DNA tatives (i.e. and Chaetomium was completely dissolved in TE buffer (10 mM Tris– globosum) the completely sequenced genomes are avail- HCl 1 mM EDTA, pH 8.0) to a final volume of 100 μL. able. Analysis of the genome of C. thermophilum [1] The concentration of obtained sample was measured in mainly focused on the presence of genes coding for Nanodrop 2000 (Thermo Scientific, Waltham, MA, nucleoporins of high thermal stability, whereas the draft USA). genome of Chaetomium globosum [2] was mainly asked for diverse genes coding cellulolytic pathways. Library preparation for DNA sequencing The filamentous fungus Chaetomium cochliodes was Approximately 1 μg of high quality genomic DNA was long considered to be a variant of Chaetomium globosum fragmented in 50 μl Low TE buffer (10 mM Tris pH 8.0, (cf. the NCBI database at www.ncbi.nlm.nih.- 0.1 mM EDTA) by BioRuptor UCD-200 sonication sys- gov/taxonomy) but already in very early studies e.g. [3] it tem (Life Technologies, Carlsbad, CA, USA) to obtain a was shown that C. cochliodes produces the antibiotic population of ~190 bp long fragments. The length and chaetomin which was shown to be highly active mainly the quantity of generated fragments were assessed by against Gram-positive bacteria. Additionally, studies Bioanalyzer chip technology (Agilent Technologies, from our laboratory revealed differences between C. glo- Santa Clara, CA, USA) according to the manufacturer’s bosum and C. cochliodes in the primary sequence and instructions. The protocol of the Library Builder™ Sys- expression profile of peroxisomal catalase-peroxidases tem (Life Technologies, Carlsbad, CA, USA) was used [4]. These findings – together with the fact that peroxi- for adaptor ligation, nick repair and fragment purifica- dases participate in diverse fungal secondary metabolism tion. The selection of 270 bp long fragments was con- pathways [5–9] – prompted us to sequence the entire ducted by the Pippin Prep instrument (Sage Science, genome of Chaetomium cochliodes strain CCM-F232 for Beverly, MA, USA) according to the manufacturer’s in- detailed comparative studies. structions. Library quantification was carried out using Here we release the draft genome of C. cochliodes, re- the TaqMan qPCR protocol of Life Technologies. construct its phylogeny and analyse the occurrence of abundant genes coding for heme containing peroxidases Genomic DNA sequencing and ORF prediction and catalases with respect to the recently described four Whole genome sequencing was carried out using the distinct heme peroxidase superfamilies [10] and the Ion Proton Technology (including the Ion AmpliSeq li- heme catalase super family [11]. Interestingly, represen- brary preparation kit, Template OT2 200 kit, Ion PI se- tatives from all five (super)families were found including quencing 200 kit, and the Ion PI chip kit version 2; Life putative bifunctional catalase-peroxidase, cytochrome c Technologies, Carlsbad, CA), according to the instruc- peroxidase, hybrid B peroxidases, cyclooxygenase-like tions of the manufacturer. A total of 34.746 Mbp, with a enzymes, dye-decolorizing peroxidases, heme thiolate median read length of 180 bp, were assembled into a draft peroxidases as well as large- and small-subunit mono- genome containing 6036 contigs (N50, 14,381). The gen- functional catalases. The occurrence of this large num- ome assembly was performed with Newbler 2.9. Genome ber and variability of genes encoding heme coverage of this sequencing was 316 x. The entire genome hydroperoxidases in C. cochliodes is discussed in com- shotgun project was deposited at GenBank under acces- parison with related fungal genomes. We support our sion LSBY00000000, BioProject PRJNA309375, BioSample genomic findings with a first round of a quantitative ex- SAMN04432217. For comparative genomic analyses of pression analysis of selected genes from all mentioned Chaetomium cochliodes genes Ensembl Fungi (http://fun- superfamilies involved in the catabolism of H2O2. gi.ensembl.org/index.html) was used. For gene prediction in sequenced C. cochliodes con- Methods tigs, HMM-based methods FGENESH and FGENESH Source and cultivation of Chaetomium cochliodes and + located at www.softberry.com [14] trained for isolation of genomic DNA closely related C. globosum & C. thermophilum were Chaetomium cochliodes CCM F-232 was obtained from used. For all peroxidase and catalase genes they were Czech Collection of Microorganisms at the Masaryk also curated manually. Zámocký et al. BMC Genomics (2016) 17:763 Page 3 of 15

Reconstruction of fungal phylogeny reconstruction MEGA 6 program suite [16] was applied Selected DNA sequence spanning the region from the 3′ on this 2474 bp long DNA alignment containing the typ- end of the 18S rDNA, the complete ITS1, 5.8S rDNA, ical fungal barcode motif [17]. Maximum likelihood ITS2 and the 5′ end of the 28S rDNA from correspond- method with 1000 bootstrap replications and general ing C. cochliodes contigs was aligned with 33 related se- time reversed substitution model were applied. Further, quences from Ascomycetes in exactly the same region uniform rates of substitutions with invariant sites and obtained from GenBank (Table 1). This DNA alignment involvement of all aligned sites with nearest-neighbour was performed with the Muscle program [15] imple- interchange and very strong branch swap filter were se- mented in MEGA 6 package with its default parameters lected as optimised parameters. The resulting tree was and 100 iterations. For subsequent phylogeny rendered 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 acutatum 1 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 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 australiensis Chaetomiaceae JQ067908 2160 Tter Thielavia terrestris Chaetomiaceae NC_016459 2233 Tasp 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 4 of 15

package. For additional verification, the same 2474 bp Agilent Technologies) with specific primers for selected long DNA alignment was subjected to phylogeny re- genes listed in Table 3. construction using MrBayes 3.2 [18]. Majority consen- sus tree was obtained from all credible topologies Results and discussion sampled by MrBayes over 200,000 generations (with a Overview of the sequenced genome of Chaetomium standard deviation of split frequencies below 0.01) by cochliodes CCM F-232 using the same GTR substitution model with gamma In total 6036 contigs were obtained from the genomic distributed rate variation across sites and a proportion DNA of C. cochliodes strain CCM F-232 deposited at of invariable sites. GenBank under accession LSBY00000000, BioProject PRJNA309375, BioSample SAMN04432217. 4141 of these contigs were larger than 500 bp. The genome size Reconstruction of molecular phylogeny of of the complete assembly was determined to be superfamilies 34,745,808 bp. This value is very near to previously de- Selected protein sequences translated from C. cochliodes termined size of closely related C. globosum (updated to contigs (Table 2B) and similar protein sequences coding 34.9 Mb) [2]. The GC content of the entire genome of for various peroxidases and catalases (i.e. hydroperoxi- C. cochliodes was estimated to 55.95 % which is a slight dases deposited at PeroxiBase http://peroxibase.toulou- difference to the corresponding value for C. globosum se.inra.fr with direct links to GenBank & UniProt) were (55.6 %). The average size of C. cochliodes large genomic aligned with the Muscle program [15] using default pa- contigs (>500 bp) in this experiment was determined as rameters and 100 iterations. Obtained alignments were 8256 bp, the N contig size was 14,381 bp and the lar- inspected and ambiguously aligned regions were ex- 50 gest assembled contig comprised 109,425 bp. As a qual- cluded from further analysis. Resulting alignments were ity control Phred quality scores were determined subjected to protein phylogeny reconstruction using according to Illumina device: the portion of Q40+ bases MEGA 6 [16] with optimized parameters according to was 34,112,976 (99.83 % of the whole genome sequence lowest Bayesian information criterion scores (Additional − draft) whereas Q39 bases portion was only 59,430 file 1: Table S1). Maximum likelihood method with 100 (0.17 %). Prediction of all possible ORFs of C. cochliodes bootstraps was chosen using the best substitution model with Chaetomia-optimised FGENESH suite [14] led in for each alignment (WAG in three cases and LG in two both DNA strands to a total value of 10,103. This count cases cf. Additional file 1: Table S1 for details), gamma is lower than the estimation for mesophilic C. globosum distribution of rates (four categories) and the presence [2] but much higher than the estimation for C. thermo- of invariant sites. Nearest-neighbour interchange was philum [1] or related thermophilic fungi. A brief com- used as heuristic method and very strong branch swap parison of three related fungal genomes is presented in filter was applied. The same protein alignments were Table 4. The average count of exons per predicted C. subjected to phylogeny reconstruction using MrBayes cochliodes gene was calculated as 3 with FGENESH. 3.2 [18]. Majority consensus tree was obtained from all credible topologies sampled by MrBayes over 500,000 Phylogeny reconstruction in the 18S r DNA – ITS1 – 5.8S r generations (with a standard deviation of split frequen- DNA – ITS2 – 28S r DNA region cies below 0.10) by using the same substitution model as First, we were interested in the exact phylogenetic pos- in MEGA. Resulting trees were rendered with FigTree ition of Chaetomium cochliodes. For this purpose we graphic suite available at http://tree.bio.ed.ac.uk/soft- have reconstructed the DNA phylogeny of its 2217 bp ware/figtree as cladograms with transformed branches. region spanning the region from the 3′ end of the 18S rDNA, the complete ITS1, 5.8S rDNA, ITS2 and the 5′ Transcriptional analysis of genes involved in peroxide end of the 28S rDNA containing the highly conserved catabolism with RT-qPCR locus described as universal fungal barcode [17]. Besides To study the level of expression of genes involved in all corresponding DNA sequences for species of the peroxide catabolism either non-induced C. cochliodes Chaetomiaceae family currently available in GenBank, samples or samples induced in the early exponential also sequences from related ascomycetous families were phase of growth with 5 mM H2O2 or 5 mM PAA (final included in this reconstruction (Table 1). The DNA align- concentration, added only for last 30 min.) were used for ment used for the phylogeny reconstruction (Additional file total RNA isolation with RNeasy Plus Mini kit (Qiagen, 2: Figure S1) reveals clear differences (i.e. substitutions, in- Netherlands). Obtained RNA samples were directly sub- sertions and deletions) in the sequence of C. cochliodes if jected to RT-qPCR assays in AriaMx6 device (Agilent compared with corresponding sequences of C. globosum in Technologies, Sancta Clara CA, USA) using the Brilliant the entire region. The phylogenetic output presented in III Ultra Fast SYBR Green Master Mix (also from Fig. 1 (obtained by two independent methods) clearly Zámocký et al. BMC Genomics (2016) 17:763 Page 5 of 15

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. Closest # Gene-superfamily relations identity* neighbour** Introns 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 oxidase (peroxisomal) CcochFeMnSOD1 0353 91 % TthFeMnSOD 2 /manganese superoxide dismutase superfamily CcochFeMnSOD2 1984 & 93 % CgFeMnSOD1 3 Iron/manganese superoxide dismutase superfamily 0805 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): 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 & 68 % Cthepox2a 1 peroxidase-catalase superfamily: Family II prob. manganese- 3438 dependent CcochhyBpox1 3712 & 100 % CghyBpox1 none peroxidase-catalase superfamily: hybrid B peroxidase 3350 CcochhyBpox2 0794 93 % CghyBpox2 1 peroxidase-catalase superfamily: hybrid B peroxidase Ccochcyox1 2133 & 83 % CgCyOx1 3 peroxidase-cyclooxygenase superfamily: cyclooxygenase 0418 Ccochlds 1074 & 91 % Cglds1 5 peroxidase-cyclooxygenase superfamily: linoleate diol synthase 4463 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 & 93 % Cgvcpo 3 non heme peroxidases: vanadium haloperoxidase 1446 Ccochgpx 0466 84 % Mthgpx 1 non-metal peroxidases: Ccoch1cysprx 1586 96 % Cg1cysprx 1 non metal peroxidases: 1-cysteine Ccoch2cysprx 1595 99 % Cg2cysprx 2 non-metal peroxidases: typical 2-cysteine peroxiredoxin CcochprxII 0388 & 95 % CgprxII 1 non-metal peroxidases: atypical 2-cysteine peroxiredoxin 1977 CcochkatA1 0438 & 94 % Cgkat1 2 heme catalase superfamily: large subunit heme catalase 2821 CcochkatA2 1883 & 87 % Cgkat2 3 heme catalase superfamily: large subunit heme catalase 2899 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 segregates Chaetomium cochliodes from closely related C. demonstrates that the thermophilic representatives (mainly elatum which is a root-colonizing fungus whose genome is C. thermophilum but also e.g. T. terrestris and M.thermo- not yet sequenced [19]. Both these fungi are separated from phila)oftheChaetomiaceae family can be considered as a sister clade represented by three different DNA sequences basal lineages of the Chaetomia clade thus suggesting that within this region coding for various C. globosum strains mesophily has evolved only secondarily in this lineage. Our with a high statistical support. This figure clearly results correlate with the previous work on thermophilic Zámocký et al. BMC Genomics (2016) 17:763 Page 6 of 15

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

fungi [20] and particularly on the thermostability of Chae- oxidoreductases producing H2O2 (Table 2A) and up tomiaceae [21] where C. cochliodes was not included at that to 20 distinct genes belonging to various heme and time. non-heme peroxidase superfamilies as well as to the heme catalase superfamily. Overview on all these Putative heme peroxidases & catalases in Chaetomium genes together with their introns composition is pre- cochliodes sented in Table 2B. All presented sequences are from Intracellular is a by- of contigs of the genome project deposited at GenBank under various physiological pathways but, unique among all accession LSBY00000000, BioProject PRJNA309375, Bio- reactive oxygen species, it serves also as an import- Sample SAMN04432217. From Table 2 it is obvious that ant signalling molecule in apoptosis and ageing [22]. genes coding H2O2 degradation exhibit a higher diversity In filamentous fungi hydrogen peroxide was shown than genes coding H2O2-releasing enzymes. Detected genes to be implicated in essential proliferation and differ- for non-heme peroxidases include vanadium-containing entiation processes [23]. Thus we have performed haloperoxidase, glutathione peroxidase as well as 1-cysteine this genomic screening for all possible ORFs coding and 2-cystein . This work focuses further on for a) enzymes supposed to release H2O2 during genes coding for heme peroxidases. their reaction and b) two main types of enzymes in- As was presented recently, there are at least four heme volved in the catabolism of hydrogen peroxide in a peroxidase superfamilies and one heme catalase superfam- novel genome of a soil Ascomycete. With TBLASTX ily that arose independently during a convergent evolution. method we could identify 8 genes for various They differ in overall fold, architecture and en- zymatic activities [10]. The following sections aim to dis- cover all genes for representatives of all five superfamilies Table 4 comparison of three related Chaetomia genomes withinthegenomeofC. cochliodes and to determine their Organism Reference Genome Comparison with Predicted exact phylogenetic positions. Heme peroxidases are found size [bp] C.coch. ORFs in all kingdoms of life and typically catalyse the one- and C. cochliodes this work 34,745,808 10.103 two-electron oxidation of a myriad of organic and inorganic C. globosum [2] 34,886,900 100.41 % 11.048 substrates. In addition to the basal peroxidatic activity dis- C. [1] 28,322,800 81.51 % 7.165 tinct families show pronounced catalase, cyclooxygenase, thermophilum chlorite dismutase or peroxygenase activities. Zámocký et al. BMC Genomics (2016) 17:763 Page 7 of 15

64/1.0

100/1.0

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98/1.0 100/1.0 99/1.0

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74

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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 sequence alignment 2,474 bp long (Additional file 2: Figure S1). Bootstrap values above 50 & posterior probabilities are shown, respectively. Scale bare represents the frequency of ML substitutions

Peroxidase-catalase superfamily HGT-event from Bacteroidetes to filamentous Ascomy- The peroxidase-catalase superfamily is currently the cetes was previously reported as a peculiarity of katG most abundant peroxidase superfamily in various gene gene family evolution [26]. Circular tree of the whole and protein databases. It is comprised of three distinct superfamily clearly demonstrates that all katG1 genes families (Families I, II and III formerly known as classes) from the Chaetomiaceae family (cf. Additional file 3: and hybrid peroxidases that represent transition forms Table S2 for abbreviations) apparently are late descen- (clades) between these families. Here we present an up- dants of this HGT event (Fig. 2 left upper part). Within dated reconstruction of the phylogeny of this largest the upper clades we observe a basal position of the known heme peroxidase superfamily analysed previously thermophilic variants from which their mesophilic coun- [24, 25]. Our updated input included already 632 terparts descended. However, a question remains complete sequences and is presented in Fig. 2. We focus whether only the coding region of katGs was transferred here on the phylogenetic positions of all representatives from bacteria to fungi or whether some neighbouring re- (ORFs) found in Chaetomia. gions were also included in such a transfer? We demon- Family I of the peroxidase-catalase superfamily typic- strate for the gene encoding KatG1 in C. cochliodes (i.e. ally contains catalase-peroxidases (KatG), ascorbate per- CcochkatG1) that the regulatory elements located on 5′ oxidases and cytochrome c peroxidases (CcP) [24]. A and 3′ regions embedding the ORF are clearly of Zámocký et al. BMC Genomics (2016) 17:763 Page 8 of 15

Cyanobacterial & Planctomycetous ancestral KatG clades

CcochKatG1 As.

As. . CgKatG1 As. MthKatG1 TterKatG1 As. CtheKatG1 As. catalase-peroxidases NeurosporaBlumeria KatG KatG clade cladePanKatG1 As. GgrKatG1 As. MagKatG1 As. hybrid B heme clades Ba. Agaricus HyBpox

CcochHyBpox1 As. CgHyBpox1sh As. MamyHyBpox As. Fusarium KatG clade As. HjHyBpox2 As. HanHyBpox1 Ba. HanHyBpox2 Ba. Aspergilli KatG1 clade As. ElaHyBpox HyspHyBpox1 HyspHyBpox3 As. peroxidases Penicillium KatG clade GprHyBpox1 Chy. As. GprHyBpox2 Chy 99/1.0 99/1.0 CuniHyBpox1 Ba. Ascomycetous KatG2s 99/1.0 81 99 CuniHyBpox2 Ba. As. 70/1.0 50 99 97/1.0 ApruHyBpox1aaHyBpox As. 56 56/1.0 V Bacteroidetes KatGs 50 99 VdaHyBpox2 As. Proteobacterial KatGs 58/0.92 BparHyBpox As. As. 99 84/1.0 65/0.85 GgrHyBpox1 As. 60 BdoHyBPox1 As. CfioHyBpox2 70 EuryarchaealBurkhodleria PssKatG1KatG KatGsclade Pb. MagHyBpox1 As. LmaHyBpox 72 PnoHyBpox1 As. As. Firmicutes KatG clade GclHyBpox1 As. Actinobacterial KatG clade 64 Bacteroidetes minor KatG clade 99 MbruHyBpox1 As. 53/0.60 55 FoHyBpox1 Firmicutes minor KatG clade 0.51 99 GmoHyBpox1 As. 0.50 HGT 99 FpsHyBpox1 As. Euryarchaeal minor KatG clade GzHyBpox1 As. 99 MperHyBpox1 Ba. 99 CfioHyBpox1 As. Proteobacterial minor KatGPtrKatG clade St. ChigHyBpox1 As. BdotHyBpox2 As. Thalassiosira KatG clade HyspHyBpox2 As. minor Stramenopilous KatG clades BfuHyBpox2 As. 99 SsclHyBpox As. CgCcP As. 97/1.0 Basidiomycetous minor hyBpox clade 59/1.0 CcochHyBpox2 As. CcochCcP As. OppiHyBpox As. CtheCcP As. 57/0.79 61 50/1.0 57 Talaromyces HyBpox1 clade As.72/1.0 MthCcP 66/0.79 MvarHyBpox1 As. NcCcP As. 77 65 92 AmonHyBpox2 As. 91 AalcHyBpox As.As. MagCcP2 99 66 99 VdaHyBpox1 GzCcP2 As. 99 99 TaspHyBpox1 As. CgraCcP1 As.As. 86 60 99 TatHyBpox1 As. 67 92 TviHyBPox1 As. AalcCcP1 62 56 As. 99 99 HjHyBpox1 cytochrome c PnoCcP1 As. 99 EfeHyBpox As. 88 BfuCcP2 99 ManiHyBpox1 As. peroxidases 98 TmiHyBpox SsclCcP2 GgrHyBpox2 As. 99 63 71 MpoaHyBpox2 As. 83 MagHyBpox2 As. Aspergilli CcP2 98 NcHyBpox1 As. Basidiomycetous CcP 76 89 NdisHyBpox1 As. 79 98 NtetHyBpox1 SmaHyBpox1 59 95 CtheHyBpox As. Saccharomycete CcP PanHyBpox1 As. MucoromyceteCsubCcP1 CcP Chl. TterHyBpox1 98/1.0 75 CgHyBpox2 85 As. Chlorophyta CcP 98 99/1.0 MthHyBpox1 As. 91 MvarHyBpox2 As. As. 82/1.0 Trichosporon HyBpox basal clade

Glomeromycete & Chytridiomycete CcP i. GprAPx-R Chy.

CgraPOX2a As. CgraPOX2a 99 98/1.0 Basidiomyc. Mn&LiPOX Fam.II As.

CfioPOX2b CfioPOX2b Dothideomycete POX2 clades As.

VdaPOX2a As. VdaPOX2a 95/1.0 PecamPOX

Aspergilli CcP1 As. GmoPOX2a Neufusicoccum POX2 clade

FoPOX2a As. FoPOX2a SchaPOX

MagPOX2 As. MagPOX2 CcochPOX2a

MEspPOX2a MEspPOX2a ChigPOX2a ChigPOX2a CthePOX2a

CgloPOX2a As. CgloPOX2a MthPOX2 CfioPOX2a MamyPOX2a Chytridiomycete CcP1 LfluPOX2b As. As. As. APx clades Glomeromycete CcP1 ChoanoflagellidaHaptophycae CcP CcP

Rhodophyta & Stramenopiles CcPAPx-CcP2 cladesMetazoan APx As. As. As.

As. As.

hybrid As. As. As. APX-CcP1 & As. ascorbate As. APx-related clades Chl.

peroxidases hybrid secretory peroxidases Family III secr. peroxidases V Fig. 2 Reconstructed phylogeny of the peroxidase-catalase superfamily with focus on newly sequenced Chaetomia ORFs. The complete tree from 632 full length 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 of taxa: Pb, Proteobacteria; As, ; Ba, ; Chy, Chytridiomycota; St, Stramenopiles; Chl, Chlorophyta; Vi, Viridiplantae eukaryotic origin (Fig. 3). In the promoter region there has evolved vertically throughout Ascomycetes. For ccp is (besides the GC box) a typical regulatory sequence – genes from both C. globosum and C. cochliodes a basal the “CCAAT” box involved in eukaryotic oxidative stress lineage represented by C. thermophilum and M. thermo- response [27]. In the 3′ untranslated region the poly-A phila is apparent in the reconstructed tree. The physio- site for corresponding mRNA formation can be pre- logical role of CcP is still under discussion. dicted with a high probability. Thus, we can conclude Further phylogenetic reconstruction of the peroxidase- that a prokaryotic katG was inserted in the fungal gen- catalase superfamily reveals that in C. cochliodes but not ome but received a typical eukaryotic transcription regu- in C. globosum a Family II representative is present lation during later evolution. The main physiological (Fig. 2 – lower part). This is very surprising for such role of KatG in C. cochliodes is most propable dismuta- closely related fungal species. However, the Family II tion of metabolically-generated hydrogen peroxide to representative from C. cochliodes has its closest neigh- molecular oxygen and water, similar to typical (mono- bour in C. thermophilum. Family II ascomycetous genes functional) catalases (see below) [24, 26]. In addition to code for hypothetical heme peroxidases with yet un- KatG Chaetomia contain genes (ccp) encoding cyto- known reaction specificity but are closely related with chrome c peroxidases (CcP, Fig. 2 – middle of the left well investigated basidiomycetous manganese and part). The relationships among the fungi presented in peroxidases (Fig. 2, labelled violet). The latter are in- the CcP phylogenetic analysis suggest that this protein volved in oxidative degradation of lignin-containing soil Zámocký et al. BMC Genomics (2016) 17:763 Page 9 of 15

CcochkatG1

S

00

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 debris and typically use Mn2+ or small organic molecules Our current reconstruction based on the phylogeny of as electron donors. selected members from the whole superfamily (compris- Additional representatives from the peroxidase-catalase ing 204 unique genes) is presented in Fig. 4. Genome superfamily in C. cochliodes include two paralogs of hybrid analysis suggests the occurrence of two representatives B heme peroxidases discovered as a new gene family only of this superfamily in Chaetomia, a cyclooxygenase-like recently [25]. Hybrid-type B peroxidases are present solely and a linoleate diol synthase. Cyclooxygenase in fungi but are related to Family III (comprised of numer- genes from C. cochliodes and C. globosum share their ous plant secretory peroxidases, labelled green in Fig. 2) closest phylogenetic neighbour (Fig. 4 upper part left) in and also to Family II (fungal secretory peroxidases men- the genome of M. mycetomatis, a human pathogenic tioned above). The basal lineage for the first paralog fungus that grows optimally at room temperature [32]. (CcochHyBpox1) together with its closely related C. globo- No cyclooxygenase genes were found in thermophilic sum counterpart appears among mesophilic Sordariomy- fungi so far. In contrast, the evolutionary reconstruction cetes (Fig. 2 upper part). The second variant of another important subfamily of Family IV, linoleate (CcochHyBpox2) containing besides the peroxidase domain diol synthases, reveals a very similar pattern for Chaeto- also an additional C-terminal WSC (sugar binding) domain miaceae as already described for the previous superfam- is not closely related with C. globosum ortholog (Fig. 2 ily. Corresponding part of the tree (Fig. 4 – upper part right). Thus, both these HyBpox paralogs are not the result right) demonstrates that genes encoding linoleate diol of a recent gene duplication but segregated rather early in synthases (lds) from thermophilic fungi (M. thermophila the evolution of fungal genomes. Transcription analysis and C. thermophilum) represent basal lineages for (Table 5 & Additional file 4: Figure S2) reveals a slight in- duction of both hyBpox genes selectively with peroxyacetic Table 5 Transcription analysis of 9 selected genes for peroxide acid in the cultivation medium. In contrast, previous results catabolism in C. cochliodes recorded with RT-qPCR. Quantitative [4]revealaconstitutivemodeofexpressionfordistantlyre- values representing relative changes of the transcription level were lated katG1 gene with hydrogen peroxide and peroxyacetic obtained by comparison of the expression of a particular gene in acid. 30 min. induced vs. non induced samples. The constitutively expressed ITS1 region was used as internal standard for Peroxidase-cyclooxygenase superfamily normalization Members of the peroxidase-cyclooxygenase superfamily Changes in expression levels against non-induced control* (comprised of Families I - VII) are widely distributed among Analysed gene Sample with 5 mM H2O2 Sample with 5 mM PAA all domains of life. In many cases they are multidomain CcochhyBpox1 1.5 x 3.0 x with one heme peroxidase domain [10, 28]. Family CcochhyBpox2 0.3 x 1.7 x IV is comprised of bifunctional cyclooxygenases possessing Ccochcyox1 0.3 x 2.3 x both peroxidase and cyclooxygenase activities. They are in- Ccochlds 0.4 x 1.8 x volved in various physiological and pathophysiological pro- cesses [29]. In mammals they are located in the luminal Ccochdyprx 3.3 x 18.5 x membrane of the endoplasmatic reticulum and mediate the Ccochhtp1 2.7 x 2.9 x conversion of free essential fatty to prostanoids by a CcochkatA2 1.1 x 0.5 x two-step process [30]. The structure and function of the CcochkatB1 0.4 x 1.1 x two distinct human paralogs (constitutive COX-1 and indu- CcochkatB2 0.6 x 1.9 x cible COX-2) were intensively investigated but a compre- * Changes in the expression levels compared to the control sample (with the hensive analysis of their diverse paralogs among eukaryotic reference value of 1.0) were calculated as relative quantities due to the microbes or even among prokaryots was only recently re- formula 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 and ported [31]. Evolutionary relationships among fungal cyclo- each inducer. Typical amplification plots and melting curves are presented in genes were not analysed in sufficient detail yet. Additional file 4: Figure S2 Zámocký et al. BMC Genomics (2016) 17:763 Page 10 of 15

As.

As.

AhspPxc1 Prb. BjaPxc1 Prb. As. As. peroxicins FspPxc1 Acb. As. ExxenCyOx CsePxc1 Prb. ExspiCyOx As. HpulCyOx AalcLDS1 As. VlongLDS1 CsubLDS As. MagLDS1 As. GgrLDS2 As. FpedCyox As. MpoaeLDS1 As. SpbrasLDS As. GzLDS1 MbruCyOx FoLDS1 As. GmoLDS1 AteCyOx1 As. PspanCyOx As. CgLDS1 As. As. As. MthLDS As. EpusCyOx2 CtheLDS As. As. As. 94 PanLDS1 As. DahapCyOx As. As. SmaLDS 100 NcLDS1 As. 88 As. 100 NtetLDS1 BdotCyOx 100 100 As. 100 BfuLDS1 MeguCyOx As. SsclLDS1 99 100 100/1.0 77 CimLDS3 As. As. StchaCyOxFoCyOx2 As. As. CposLDS3 66 100 AcapLDS3 As. 59 100/1.0 BebasCyOx As. 100 As. AflLDS3 As. As. 100 100 AorLDS3 As. StchlCyOx 100 81/0.98 AfumLDS2 74 100 BiochCyOx As. 99 NfLDS2 As. As. AunamCyOx As. 83 100 52 62 AteLDS4 100 91 AusubCyOx As. As. 100/0.92 AfumLDS3 As. 100 NfLDS3 100 As. AupulCyOx 100/0.97 AclLDS3 As. As. AumelCyOx As. 99 100 AniLDS3 As. AnLDS3 NtetCyOx As. 100 100 As. 76 AteLDS3 As. 76 96 AflLDS4 As. As. MamycCyOxNcCyOx As. As. 94 AorLDS4 As. 71 65 CzmLDS1 CgCyOx1 As. 89 100 100/1.0 100 100 99 67 95 PnoLDS1 As. 100 98 100 BcomCyOx CimLDS1 As. CposLDS1 100 AbraCyOx As. 97 AcapLDS As. As. 88/1.068/1.0 100/0.53 RrufCyOx 100/1.0 AnLDS1 As. As. PfijCyOx As.As. AniLDS1 CzmCyOx As. 100/1.0 AteLDS1 100 AflLDS1 As. PficCyOx As. 100 AorLDS1 As. EpusCyOx1 As. 99 95 100 100 96 AclLDS1As. 78 100 65 As. CorbCyOx As. AfumLDS1 68 NfLDS1 As. CgloCyOx As. 62 100 As. 90 100 SsclLDS2 NhaeCyOx As. 56 100 GgrLDS1 As. GzCyOx1 As. 100 96 MagLDS2 As. GmoCyOx1 58 100 As. 85 CyegLDS As. GfujiCyOx As. 99 98 86 92 AclLDS2 As. 100 FoCyOx1 As. 59 100 AniLDS2 As. 100 MimmunCyOx Ac. 100 100 AorLDS5 As. 88 McheloCyox Ac. 59 89 56 CimLDS2 As. 100 CposLDS2 MabscCyOx Ac. 86 As. Ac. 100 100 GzLDS3 As. NoastCyOx Ac. 81 NcLDS2 As. GorhiCyOx 100 GmoLDS3 100 100 60 98 As. MneworlCyOx Ac.Ac. GmoLDS2 100 As. GospuCyox 97 97 GzLDS2 As. 98 100 100 MuflCyOx Ac. 60 72 AalcLDS2 100 AflLDS2 As. 100 FspCyOx1 Ac. AorLDS2 As. Ac. 59 SaceryxCyox Ac. 97 100 100 AnLDS2 As. Ac. StfulCyOx 100 100 92 AteLDS2 As. 82 RhdelLDS Mu. AmvanCyOx 62 100 RhmicLDS Mu. AmmethCyOx Ac.Ac. 100 100 PaparLDS Mu. MvulCyOx Ac. 100 Ac. 100 MambLDS Mu. MfortCyOx McirLDS anMu. 54 100 LmacLDS MsmCyOx1 87 90 100 ScelCyOx Dp. 100 PnoLDS2 As. Ap. 70 100 SreLDS Ba. MxCyOx1 Dp. . 88 100 100 As. 85 100 UmLDS1 Ba. SuCyOx Aci. . 96 77 51 100 55 SrosLDS1 Ba. . 100 BrspCyOx 83 SrosLDS2 Ba. . 60 100 92 MlpLDS1 Ba. . CalspCyOx Cy peroxidockerins 73 MlpLDS2 Ba. . 100 98 MlpLDS3 Ba. LepspCyOx Cy 100 . 92 CyspCyOx Cy 79 PcLDS2 Ba. 100 . 99 PplLDS2 Ba. 100 100 AcmaCyOx Cy . 100 JargLDS2 Ba. olspCyOx Cy 100 T 99 GluxLDS2 Ba. 100 100 100 CcinLDS2 Ba. LirobCyOx Cy 100 59

77 AbLDS2 Ba. 52 100 LbiLDS2 Ba. MprodCyOx Cy 87 MchtCyOx Cy PinvLDS2 Ba. cyclooxygenases 97 SbreLDS2 Ba. lionoleate diol SlutLDS2 Ba. 55 73

70

100 ScomLDS2 Ba. 76 99 CochthCyOxAeuCyOx2 Cy St. PindLDS2 Ba. AeuCyOx1St. PindLDS1 Ba. SbreLDS1 Ba. 100 97 SlutLDS1 Ba. synthases PinvLDS1 Ba. PcLDS1 Ba. BmaPxDo1 PlB. AbLDS1 Ba. GluxLDS1 Ba. ScomLDS1 Ba. CcinLDS1 Ba. LbiLDS1 Ba. JargLDS1 Ba. PplLDS1A ACmaPxDoCintPGHS2 Cyb. De. PplLDS1B Ba. CintPGHS1GfrCyOx2 De. Cn. .

a GfrCyOx1 Cn.

B HsPGHS1 De.

BtPGHS1 De. 3

S

CfaPGHS1 De. D EcabPGHS1 De. Ba. DrPGHS1 De. L GgaPGHS1 De.

FhePGHS De. g DrPGHS2 De.

r PcLDS3 Ba. HsPGHS2 De. BtPGHS2 De. a GgaPGHS2 De. JargLDS4 Ba. J

CpoPGHS2 De. CfaPGHS2 De.

EcabPGHS2 De. Fig. 4 Reconstructed phylogeny of the peroxidase-cyclooxygenase superfamily with focus on Chaetomia ORFs. The complete tree from 204 full length 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 corresponding genes in mesophilic Chaetomia. Only re- decolorizing peroxidases (DyPs). These heme enzymes cently it was shown that fatty acid diol synthases are were first isolated from soil basidiomycetes but were fur- unique fusion proteins containing a N-terminal heme ther shown to be present in a wide variety of fungi and peroxidase domain joined with a C-terminal P450-heme bacteria [35]. DyPs catalyse the H2O2-mediated oxida- thiolate domain for conversion of unsaturated fatty acids tion of a very broad range. Originally, fungal to dihydroxy-fatty acids [33]. These enzymes are an es- representatives were found to degrade bulky dyes. A de- sential part of the psi factor sexual inducer cascade in tailed structure- and sequence-based comparison dem- various fungi [34]. Their exact physiological role within onstrated that DyPs together with chlorite dismutases the life cycle of Chaetomiaceae needs to be elucidated in and chlorite-dismutase like proteins (EfeB, HemQ) con- the future. Our first round of transcription analysis re- stitute the CDE superfamily [36], also designated as vealed around 2-fold induction of expression of both peroxidase-chlorite dismutase superfamily [10]. The re- cyox1 and lds genes in a medium with peroxyacetic acid constructed evolution of DyPs within this superfamily is (Table 5 and Additional file 4: Figure S2). shown in Fig. 5. In fungal genomes mainly representa- tives of the subfamilies DyP-type D and DyP-type B can Peroxidase-chlorite dismutase superfamily be found as paralogs. Interestingly, in the genome of C. Our next screening within the C. cochliodes genome fo- cochliodes only a fused version of DyP-PFL is present, cused on the presence of genes encoding dye- i.e. an N-terminal DyP peroxidase domain connected Zámocký et al. BMC Genomics (2016) 17:763 Page 11 of 15

clades of Cld & CldL (bacterial & archaeal)

CcochDyPrx As. HaantDyPrxBfDyPrx2 Pb. Deu. GmoDyPrx2

GfujiDyPrx

fusions DyP-PFL cuDyPrx1 Ba.

BaDyPrx3 Ba. alDyPrx Ba. T PbreDyPrx3 Ba. PolypPrx Ba. T CgDyPrx PoDyPrx1 Ba. PinDyPrx As. DespDyPrx Pb. AdelDyPrx7 Ba. AaurDyPrx Ba. JaagaDyPrx Pb. ExglaDyPrx Ba. As. MperDyPrx2 Ba.

CollareDyPrx Pb. 99 MscDyPrx1 Ba. As. 99 GluxDyPrx7 Ba. As.

CocorDyPrx Pb. 72 CcinDyPrx1 Ba.

74 AmusDyPrx1 Ba. As.

99 GlucDypPrx1 Ba. CviolDyPrx Pb. 99 AcicalcDyPrx Pb. 99/1.0 77 GsppDyPrx2 Ba. 75 60 MlpDyprx1 Ba. 98 As. 99/1.0 RsoDyPrx Pb. 94/1.0 AflDyPrx As. 99 AorDyPrx As. ThbiDyPrx Ac. 97 As. 99/1.0 AfumDyPrx As. 95 99 MispDyPrx Ac. NfDyPrx As. RhospDyPrx1 Ac. 93 92 PechrDyPrx2 99 99 GobronDyPrx AclDyPrx2 99 99 As. 53 CzmDyPrx HGT As. BxDyPrx1 Pb. 55 99 BysspDyPrx As. PaespDyPrx Fi. Ac. 91 SsclDyPrx As. 99 PcurDyPrx Fi. SborDyPrx 89 BfuDyPrx As. 73 BsDyPrx Fi. 91 NcDyPrx As. AbdeDyPrx Fi. 96 SmaDyPrx 68 99 99 97 StheCldL 99 SmaDyPrx2 As. 96 SsaDyPrx Fi. 99 99 LbiDyprx2 Ba. . Fi. 88 99 PplDyPrx1 Ba. LhofDyPrx Fb. 99 99 NospuDyprx Cy GesaDyPrx Fi. 79 99 AcmaDyPrx Cy. TcelDyPrx 95 RbaDyPrx Pmc. 90 98 Ac. 85 TfuDyPrx Ac. AcspDyPrx Ac.Ac. 99 99 NfaDyPrx1 AcmiDyPrx Ac. MeteyDyPrx Pb. MvaDyPrx1 86 Ac. 82 98 97 RhlegDyPrx Pb. MsmDyPrx1 Ac. 99 98 74 SlingDyPrx Bi. SthygDyprx Ac. 99 AvarDyPrx Cy. CaacDyPrx Ac. 99 RbrooDyPrx Cy. RpDyPrx Pb. 95 99 CyanspDyPrx2 Cy. 94 99 OantDyPrx2 Pb. 67 CyanspDyprx3 Cy. YpDyPrx1 Pb. 99 52 CycmarDyPrx Bi. 57 KpnDyPrx Pb. 99 ChDyprx Bi. 99 NeDyPrx Pb. CityoPrx Pb. 90 82 99 NimulDyPrx Pb. SboDyPrx1 Pb. 99 MoprDyPrx Cy EcEfeB Pb. . As. MealFusDyPrx Pb. PEchDyPrx1 63 CyanspDyPrx1 Cy. 98 99 99 VniDyPrx Pb. HpulDyPrx As.As. 58 V OmaiDyPrx ibnigDyPrx Pb. 71 AcspDyPrx Aci. ByspecDyprx As.As. 99 99 67 PpacDyPrx Pb. AorDyPrx1 As. 99 OantDyPrx Pb. As. 81 AflDyPrx1 MopeDyPrx Ac. 94 92 TminDyPrx 99 75 MiluDyPrx 99 59 60 As. 99 88 AmymedDyPrx CacorDyPrx As. 93 89 DraDyPrx Dei. Ac. ClpsDyPrx 99 As. 99 NocaDyPrx 98 MacrDyPrx As. As. SsvDyPrx Ac. 99 ManiDyPrx 99 CgleDyPrx Bi. Ac. CgloDyPrx As. 99 PsspDyPrx Pb. Ac. 99 As. 99 96 AclwDyPrx Pb. NhaeDyPrx As. EnaeDyPrx Pb. 62 KoverDyPrx GmoDyPrx1FoDyPrx 99 99 CalspDyPrx Cy. 58 MsmeDyPrx Ac. PmDyPrx As. 99 89 56 68 MYspDyPrx3 Ac. GzDyPrx As. 99 SavDyprx3 Ac. 99 91 Aci. Ac. 99 KteracDyprx Chl. MlaeDyPrx Ac. 54 99 FrspDyPrx asDyPrx Ba. 63 T 87 92 67 57 SusDyPrx 99 SocDyPrx Pb. RjoDyPrx Ac. 99

99 97 CyfusDyPrx Pb. MxDyPrx Pb. 78 dye-decolorizing 99 CorcorDyPrx Pb.

99 PaninDyPrx vir

ZmoDyPrx Pb. 81 RhospDyPrx2 PandulDyPrx vir. Ac. CtaiDyPrx Pb. PansalDyPrx vir Aci. AccasDyPrx CtesDyPrx1 Pb. PputDyPrx Pb. PfDyPrx2 Pb. peroxidases (DyPs) CcrvDyPrx Pb.

BfrDyPrx Bi. .

CconDyPrx Pb. l re.

PdiDyPrx Bi. o

T Tre.

Alv.

M

BtheDyPrx Bi. Amb.

x r

AcspDyPrxSonDyPrx Pb. Pb. . P

Amb. y

ibcholDyPrx Pb. D .

DdDyPrx Myc. l

V ViblenDyPrx Pb. EcDyPrx2 Pb. u

SflDyPrx2 Pb. v BfDyPrx1 Deu.

PtetDyPrx r

GalanaDyPrx Pb. A

NgrDyPrx2 Amb.

NgrDyPrx1 SchjapDyPrx SchmanDyPrx SchmanDyPrx Fig. 5 Reconstructed phylogeny of the peroxidase-dismutase superfamily with focus on newly discovered Chaetomia sequences forming a separate clade of DyP-Bs together with fused bacterial representatives from which they were derived by a HGT event. The complete tree from 282 full length sequences is presented with 655 sites aligned. C. cochliodes sequence is labelled red. Distinct subfamilies are labelled in different colours. Values in nodes represent bootstrap values above 50 (from maximum likelihood analysis) and posterior probabilities (from Mr. Bayes), respectively. Abbreviations of peroxidase names are 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

with a C-terminal pyruvate formate- (PFL) domain components necessary for the glycyl formation known as a glycyl radical containing region [37]. This with yet unknown physiological function. A HGT event unique gene fusion was detected also in other distantly with a high bootstrap support in the clade of fused DyPs related prokaryotic & eukaryotic genomes [38]. The PFL B can be observed between proteobacteria and ascomy- domain can be activated by PFL activase, a radical SAM cetous fungi (Fig. 5 and Additional file 6: Table S4 for superfamily member [39], but the significance of a PFL abbreviations). As the fused DyP B-PFL proteins are yet fusion with a peroxidase domain remains elusive. We hypothetical, their physiological relevance has to be de- could detect a putative PFL activase in C. cochliodes termined among Chaetomiaceae. Our first round of contig 00230 revealing 81 % identity with CHGG_03160 transcription analysis of dyprx gene exhibited the highest from C. globosum and other putative PFL activases from induction observed among all 5 superfamilies followed filamentous fungi. Thus, C. cochliodes possesses both in this study with hydrogen peroxide (3-fold) and mainly Zámocký et al. BMC Genomics (2016) 17:763 Page 12 of 15

with peroxyacetic acid (18.5-fold) in the cultivation paralogs is variable (Fig. 6). Whereas there is a thermophilic medium (Table 5). basal lineage for CcochHTP2 and CcochHTP3 and their corresponding counterparts in C. globosum,thesituation Peroxidase-peroxygenase superfamily for paralog CcochHTP1 is different. Corresponding genes Heme-thiolate peroxidases from Fungi and Stramenopiles from pathogenic fungi represent a basal lineage for closely constitute the peroxidase-peroxygenase superfamily [10]. related CcochHTP1 and CgHTP1. It is unknown so far Enzymes encoded by htp genes represent probably the whether these three putative heme-thiolate peroxidases ex- most versatile catalysts among peroxidase superfamilies hibit different enzymatic properties but they were segre- thus catalysing on one side classical heme peroxidase reac- gated early during the evolution of fungal genomes and tions and on the other side monooxygenase (monohydroxy- thus they all may be interesting for biotechnological appli- lation) reactions like cytochrome P450s [40]. The cations. We have also performed transcription analysis of reconstructedphylogenetictreefortheperoxidase- htp1 gene paralog resulting in almost 3-fold induction both peroxygenase superfamily (Fig. 6) reveals the distribution of with hydrogen peroxide and peroxyacetic acid present in three gene paralogs of this superfamily within the Chaeto- the cultivation medium (Table 5). mium cochliodes genome. The presence of multiple gene paralogs in genomes of ascomycetous fungi is frequent and Putative heme catalases in Chaetomia occurred by repeated gene duplications of this rather short Typical (monofunctional) heme catalases are enzymes gene but the phylogenetic distribution of C. cochliodes that very efficiently dismutase hydrogen peroxide to

GmargHTP1 Ba. As. - basidiomycetous 2 As.

LbiHTP5 Ba. P

T As. As.

AaeAPO1 2yorBa. AbHTP4 Ba. H

c AbHTP3 Ba. i AaeAPO2 Ba. f As. AnHTP1 P As. As. OmaiHTP As. TterHTP1 As. peroxidase- StcharHTP As. StchlHTP CgHTP2 As. As. CcochHTP2 As. As. AbHTP5 Ba. CopradAPO1CcinHTP4 Ba. Ba. CgHTP3 MthHTP As. As. ThhetHTP1 peroxygenases ElaHTP AdelHTP5 Ba. 67 AorHTP1 As. As. PbraHTP1 As. 100 AdelHTP1 Ba. 56 PnoHTP2 (HTPs) DsepHTP1 UmHTP2 Ba. SpmusHTP 100 100/1.0 AniHTP3 UmHTP1 Ba. 71 100/1.0 53 As. 96 BcinHTP1 - ascomycetous SreHTP2 Ba. 83 72/0.55 SsclHTP1 As. As. PnoHTP6 CcochHTP1 As. 100/1.0 PnoHTP1 CgHTP1 100 100 MamyHTP1 As. PnoHTP8 As. 71 As. As. MpoaeHTP 1 As. PanHTP1 As. 99/1.0 PnoHTP3 As. PnoHTP12 78 92 AniHTP2 100 89/1.0 PnoHTP10 As. AteHTP2 As. As. 93 As. 68 AorHTP2 As. As. MpiHTP1 As. 100/1.0 100 AparaHTP As. PnoHTP9 As. 100 PnoHTP1 As. 100 GzHTP5 96 86 AniHTP1 97 60 As. 75 93/0.8 PficHTP1 GzHTP4 As. 55 PnoHTP13 79/0.54 AteHTP1 - ascomycetous As. GzHTP1 As. AteHTP3 As. 100 As. 50 67 ColfioHTP As. AfumHTP2 As.As. 65 As. 65 86 96 ChigHTP2 AorHTP4 As. 77 ColgloeoHTP 99 AfumHTP3 As. 100 ChigHTP1 As. 100 SlutHTP5 Ba. 77 66 GfujHTP1 As. 100 100 SbreHTP5 Ba. GmoHTP1 As. 99 88 As. GzHTP2 SlutHTP1 Ba. 72 75 As. PangHTP4 SbreHTP1 Ba. 79 As. 54 NcHTP1 100 100 SlutHTP2 Ba. MagHTP1 As. SbreHTP2 Ba. 67 60/0.89 As. 100 AniHTP5 SerlaHTP1 Ba. 100/1.0 CcochHTP3 As. 100/1.0 PinvHTP2 Ba. 56 CgHTP4 As. 100/0.97 98 77/0.79 T PinvHTP1 Ba. islHTP1 As. 100 TterHTP2 As. LbiHTP2 Ba. 60 91 MroUPO1 Ba. LbiHTP1 Ba. 100 MrorHTP4 Ba. 64 AbHTP1 Ba. 100 GluxHTP1 Ba. 89 CcinHTP2 Ba. 100 GluxHTP3 Ba. 99 73 GluxHTP2 Ba. CcinHTP1 Ba. 88 CytorHTP1 Ba. DsquaHTP1 Ba. 100 46 HirHTP1 Ba. 94 MrorHTP1 Ba. 100 MrorHTP5 Ba. PcHTP1 Ba. 100 MrorHTP6 Ba. 73 - basidiomycetous PcarHTP1 Ba. 100 MperHTP1 Ba. PplHTP2 Ba. 62 MrorHTP2 Ba. PplHTP3 Ba. MrorHTP3 Ba. As. PplHTP1 Ba. 99 100 PnoHTP4 As. 100 AniHTP4a PnoHTP7 100 100 AniHTP4b As. CsubHTP1 Ba. 93 PnoHTP5 As. As. 69 76 HminHTP1 As. 98 100 DacspHTP1 Ba. 70 MellpHTP Ba. OpsiHTP1 As. 67 98 80 100 OpsiHTP2 94 100 MlarHTP1 Ba. 100 82 100 CfumagHTP1 As. UhHTP1 Ba. 100 98 100 CfumagHTP2

82 HjHTP1

98 As. - basidiomycetous PsanHTP1 Ba. 91 SreHTP1 Ba. TharHTP1 AphasHTP - ascomycetous

UmHTP3 Ba. 100 90 AphinHTP2 St. 100 As. 51 100 SdicHTP1 St. As. PhubHTP1 Ba. 100 AphinHTP1 St. RirHTP1 Mu. As. 92 AkawHTP1 100 100 97 AorHTP3 As. St. 59 PparHTP2PiHTP1 St. St. 100 AfumHTP1 100 PoxalHTP1

78 GzHTP3 NhaeHTP PramHTP2 St. PsojHTP3 St. MuccirHTP RdelHTP1 Mu. RorHTP1 Mu. As. PparHTP3PiHTP2 St. St.

As. . . PulHTP2 St. As. As.

a

a As.

As. B B PsojHTP2PulHTP1 St. St. As.

4 3

P PsojHTP1 St. P Mu.

T PparHTP1 St. T

H H - oomycetous PramHTP1 St. Ba. AbHTP2

r

c LbiHTP4 Ba. LbiHTP4

a SAcoHTP2 SlutHTP4 Ba. LbiHTP3 Ba.

PcHTP2 Ba. P

SAcoHTP1 PplHTP5 Ba. PinvHTP4 Ba. Ba. PplHTP4 c

CcinHTP3 Ba. CcinHTP3 PinvHTP3 Ba. PinvHTP3

P - mucoromycetous 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 subfamilies are 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 13 of 15

oxygen and water. In contrast with heme peroxidases and their C. globosum counterparts, a situation very they can both reduce and oxidize hydrogen peroxide and similar to the peroxidase superfamilies. In contrast, have negligible peroxidatic activity [41]. Heme catalases there are mesophilic basal lineages for the small sub- represent a monophyletic group that evolved as a dis- unit peroxisomal catalases CcochKatB1 and Ccoch- tinct gene family from to almost all lineages KatB2 (Fig. 7 – on the right). In particular, of eukaryotes [11]. In Fig. 7 the phylogeny focused on CcochKatB1 and CgKatB1 have a basal lineage among fungal heme catalases is presented. There are 3 distinct catalases from various soil and phytopathogenic fungi. clades of genes for typical catalases defined by Klotz et Surprisingly, CcochKatB2 has no counterpart in the al. [42]. In fungi only representatives of Clade 2 (large closely related genome of C. globosum. Putative cata- subunit, secretory catalases) and Clade 3 (small subunit, lase from a widely distributed soil fungus S. schenckii mostly peroxisomal catalases) can be found. There are shares a common ancestor with this unique small up to four gene paralogs of a catalase gene within C. subunit peroxisomal catalase of C. cochliodes (Fig. 7). cochliodes genome that underlines the importance of Possible involvement of C. cochliodes four catalase mostly monofunctional catalases for the removal of isozymes in the defence against oxidative stress was H2O2. There are thermophilic basal lineages for the large analysed by RT-PCR. Obtained results in the early ex- subunit secretory catalases CcochKatA1, CcochKatA2 ponential phase of fungal growth show only a slight

.

t

-catalase- VvKatFus Eudi. D

OsjapFus Mctd. 1 SbiKatFus Mctd. t ACmaKatLox1 Cy SmKatFus Cryp. RcFus Eudi. a

NOspKatLox1 Cy K

fusion o

r PhoKatLox1 Cn. t HsKatA Dt. HsKatA P PabeKat1 Dt. CajaKat1 Dt. BtKatA Dt. CfaKat1 Dt. SscKat1 Dt. MmKat1 Dt. XlKat2 Dt. CvirKatLox1GfrKatLox Cn. Cn. XtKat1 Dt. XlKat1 Dt. KmaKat1 Dt. DrKat1 Dt. HmolKat1 Dt.

DYfeKat1 Bi. 94 DmaKat1 Ec. PEspKat1 Bi. PvanKat1 Ec. AviKat1adKat1 Cn Pl. . T CHpiKat1 Bi. . 98 99 100 AaeKat1 Ec. AaeKat2 Ec. PEheKat1 Bi. 100 66 100 AgaKat1 Ec. FjKat1 Bi. 100 99 BmorKat1 Ec. 99 SpliKat1 Bi. DpKat1 Ec. CHglKat1 Bi. 100 DmKatE1 Ec. Ap. . 100 HcoKat1 Ec. 93 . CelKat1 Ec. FbKat1 Bi. 69 100 CbrKat1 Ec. CatKat1 Bi. 100 63 POsKat1 Pb. 100 TgKat1 98 RoKat1 Zy catalases VpaKat1 Pb. 100 95 80 PblKat1 Zy 100 BpKat1 Pb. 70 NgrKat1 Hl. BphyKat1 Pb. 100 100 100 PtetKat1 Ci. TthKat1 Ci. 62 PfKat3 Pb. 100 CowKat1 Ich. . PpuKat3 Pb. 100 100 PpalKat1 My. . BceKat1 Fi. DdKat1 My. Ar 100 100 . BanKat1 Fi. DpuKat1 My 81 EXsKat1 Fi. MarKat1 Ar. BdeKat2 Chy. 100 56 MbaKat1 Ar 99 59 100 44 100 100 72 MbuKat1 OiKat Fi. BsKat1a Fi. MEmaKat1 Ar. 100 74 84 100 BsKat1b Fi. MmazKat1 100 63 KraKat1 Chl. 86 BalKat1 Fi. 100 MpaKat1 Bi. Ar. 100 BsKat2a Fi. PpuKat1 Pb. 98 PpuKat2 Pb. BhaKat1 Fi. 100 97 100 PfKat1 Pb. . DalKat1 Fi. SgKat1 Pb. FspKat1 Ac. 90 100 57 NOpuKatE Cy 97 NfaKatB Ac. 52 RmaKat Bi. MavKat1 98 CjejKat1 Pb. Ac. 100 46 SerKat2 SmeKat1 Pb. Ac. 80 STroKat1 Ac. 53 HaurKat1 Chl. 100 PproKatE Pb. SsvKat1 67 Ac. VpKat1 Pb. SscaKat1 Ac. 60 100 70 PaerKat1 Pb. AkmKat1 Ver. 100 100 79 81 SmKat3a Cryp. LAinKat1 Pb. PpaKat5 Cryp. SetyKatE Pb. 100 PpaKat3 Cryp. 87 100 EcoKatE Pb. 100 74 PpaKat4 Cryp. LplKat1 Fi. DpuKat2 My. 91 DdKat2 My. 100 86 SerKat1 Ac. 99 CgluKatE CnKat1 Ba. 100 Ac. 100 CjeKatE 60 100/1.0 Ac. PoKat2 Ba. 92 1.0 CgaKat1 Ba. As. 97 100 67/0.93100/1.0 AorKat1 95 CnKat4 Ba. 100 77 74 AfumKat1 As. ArolKat1 As. 100 As. AnKat1 100 80 CcochKatB2 As. 100 SschKat1 AniKat1 As.As. 91 NcKat4 As. CimKat1 As. 100 65 89 100 GzKat2 As. AcapKat1 100/1.0 99 100 GmoKat1As. As. SsclKat1 As.As. 97 53/1.0 PechKatE1 As. BfuKat1 As. 91 61 100/1.0 TorhemKat1 As. PtritKat1 100 ScboKat1 As. 100/1.0 CheKat2 HarKat1 As. As. 89 95 100 CcochKatB1 PnoKat2 As. 93 98 99 81 100 CgKatB1 As.As.

CgraKat1 59 As. 100 75 DhKat1 As. As. 100 PguKat1 As. NhaeKatE1GzKat1 As. 51 50/1.0 SceKatT As. KlKat1 53 As. MagKatE1 50 100 100 YlKat1 CfulKat2 As.As. 1.0 58 60 53/0.66 PgrKat1 Ba.As. -Clade 3 small subunit, CtherKat1NcKat1 As. GzKat4 As. 100/1.0 97 AniKat2 PanKatA As. 100 100 PEchKatC As. 100 88/0.72 As. 55 CgloKat1 peroxisomal As. MamyKat1 As. 100 SpomKat1 As. TterKat1 As. 100 100 CboiKatA MthKat1 As. 51 AgoKat1 67 100 CgKatA1 81 SceKatA As. 90 64 100 CalKat1 As. 100 PstiKat1 As. As. As. 100 As. 92 100 PcKat3 Ba. SmeKat2 Pb. As. 62/0.72 As. PblKat2 Zy. CcinKat4 Ba. As. 82 As. CcochKatA1 As.RoKat2 Mu. 61 100 CtherKat3 CheKat1 As. As. 97/1.0 83 PnoKat1 99 PEchKat3AnKatB 100/1.0 100 BhaKat2 Fi. As. 100 BsKat2b Fi. 99/1.0 BdeKat1 Chy PnoKat3 As. 51 50 PfKat2 Pb. As. ZmKat3 Mctd. 100 SbKat1 Mctd. 100 As.

MagKatE2 As. As. OsKat1 Mctd.

CpurKat2GzKat3 As. Eudi. PtKat1 AtKat3 Eudi. As.

GhKat1 Eudc. GhKat1 CreKat1 Chlph.

PpeKat1 Eudi. PpeKat1 PpaKat1 Cryp.

PdelKat2 Eudi. PdelKat2 PpaKat2 Cryp.

AlyKat2 Eudi. AlyKat2 ZmKat2 Mctd. AtKat02 Eudi. AtKat02 SmKat1a Cryp.

NcKat3 SmKat01b Cryp.

CgraKat2 Eudi. NtKat1 AcvKat2 Cryp.

As. Eudi. AtKat1 ZaKat1 Mctd. ZaKat1 MpKat1 Cryp.

ZmKat1 Mctd. ZmKat1 AtrKat1 BMagno. -Clade 2 large subunit, OsKat2 Eudi. A As.

PanKatB t r

K CtherKat2 As. . a

SctheKat1 t secretory 2 MthKat2 As. MamyKat2ThhetKat1 As. As. CgKatA2 B M -Clade 1 small subunit

a

g

n

o CcochKatA2 . Fig. 7 Reconstructed phylogeny of the heme catalase super family with focus on Clade 2 and 3 representing the distribution of Ascomycetous large subunit as well as small subunit catalases (labelled in different colors). The complete tree from 222 full length sequences is presented with 546 sites aligned. C. cochliodes paralogs are labelled red. Distinct clades are labelled in different colours. Values in nodes represent bootstrap values above 50 (from maximum likelihood analysis) and posterior probabilities (from Mr. Bayes), respectively. Abbreviations of peroxidase names are 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 Zámocký et al. BMC Genomics (2016) 17:763 Page 14 of 15

induction of the paralog katB2 in the medium con- Acknowledgements taining peroxyacetic acid (Table 5). Our research was supported by the Austrian Science Fund (FWF, project P27474-B22), by the Slovak Grant Agency VEGA (grant 2/0021/14) and by the Slovak Research and Development Agency (grant APVV-14-0375). We thank Conclusions the company Hermes LabSystems for providing us with AriaMx6 device. In conlusion genomic sequence analysis revealed that Availability of data and materials Chaetomium cochliodes is closely related to C. globosum & All used DNA sequences are deposited in GenBank (Table 1). All protein C. elatum. These three filamentous fungi are mesophilic sequences that were used for reconstruction of phylogenies are listed in but probably have thermophilic ancestors as revealed from Additional file 3: Table S2, Additional file 6: Table S4, Additional file 5: Table S3, Additional file 7: Table S5 and Additional file 8: Table S6. If possible their their basal lineage. C. cochliodes contains heme peroxidases PeroxiBase accession number is given to find them at (http:// and catalases from all so far described superfamilies. Asco- peroxibase.toulouse.inra.fr) if no PeroxiBase accession numbers exist yet their mycetous genes encoding catalase-peroxidase and dye de- UniProt (http://www.uniprot.org) accession numbers are given. colorizing peroxidase were obtained during the evolution ’ by horizontal gene transfer from various bacteria. Several Authors contributions MZ selected the fungus, designed all experiments, performed all molecular heme peroxidases of Chaetomia like hybrid heme B perox- phylogeny analyses and wrote the manuscript; AK cultivated the fungus and idase, linoleate diol synthase or DyP-type B form fusions performed genomic & transcription analyses; KC optimised the isolation of with additional functional domains that might enable a fungal DNA and performed genomic & transcription analysis; KL prepared the genomic DNA for sequencing and contributed to the discussion; HT broader catalytic variability. Furthermore cytochrome c per- performed the sequencing and assembled the contigs; CO evaluated the oxidase, manganese and three paralogs of heme-thiolate classification and phylogeny of peroxidases & catalases and finalized the peroxidases are found in addition to typical (monofunc- manuscript. All authors read and approved the final manuscript. tional) catalases of large and small subunit architecture. Competing interests Our transcription analysis reveals the highest induction of a The authors’ declare that they have no competing interests. fused dyprx gene with hydrogen peroxide and mainly with peroxyacetic acid in the cultivation medium followed by Consent to publish moderate inductions of htp1 and hyBpox1 genes. Not applicable (this manuscript does not contain any individual persons data).

Additional files Ethics approval and consent to participate Not applicable for this fungal genomic study. None of here analysed genes Additional file 1: Table S1. Substitution models with the lowest of Chaetomia was used in experimental cloning research (yet). Bayesian information criterion scores for all 5 superfamilies analysed in this contribution. (XLSX 70 kb) Author details 1Department of Chemistry, Division of Biochemistry, University of Natural Additional file 2: Figure S1. DNA sequence alignment of genomic DNA Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. 2Institute from 34 ascomycetous fungi in the region covering 18S rDNA – ITS1 – 5.8S of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, rDNA – ITS2 – 28S rDNA (fasta format). (FAS 85 kb) SK-84551 Bratislava, Slovakia. 3Department of Biotechnology, University of Additional file 3: Table S2. Abbreviations of peroxidase gene names Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria. used for the peroxidase-catalase superfamily. (XLSX 54 kb) Additional file 4: Figure S2. A typical profile of real-time quantitative Received: 16 August 2016 Accepted: 22 September 2016 PCR analysis of transcripts from peroxidase genes obtained from C. cochliodes under oxidative stress. Upper panel: amplification plots for hyBpox1, cyox1andlds genes detected with SYBR Green Master Mix (Agilent References Technologies). Lower panel: melting curves for hyBpox1, cyox1andlds genes 1. Amlacher S, Sarges P, Flemming D, van Noort V, Kunze R, Devos DP, presented in Table 5. (TIF 450 kb) Arumugam M, Bork P, Hurt E. Insight into structure and assembly of the Additional file 5: Table S3. Abbreviations of peroxidase gene names nuclear pore complex by utilizing the genome of a eukaryotic thermophile. used for the peroxidase-cyclooxygenase superfamily. (XLSX 27 kb) Cell. 2011;146:277–89. 2. Cuomo CA, Untereiner WA, Ma LJ, Grabherr M, Birren BW. Draft genome Additional file 6: Table S4. Abbreviations of peroxidase gene names sequence of the cellulolytic fungus Chaetomium globosum. Genome used for the peroxidase-dismutase superfamily. (XLSX 23 kb) Announc. 2015;3:e00021–00015. Additional file 7: Table S5. Abbreviations of peroxidase gene names 3. Geiger WB, Conn JE, Waksman SA. Chaetomin, a new antibiotic substance used for the peroxidase-peroxygenase superfamily. (XLSX 18 kb) produced by Chaetomium cochliodes. J Bacteriol. 1944;48:527–36. Additional file 8: Table S6. Abbreviations of catalase gene names used 4. Zámocký M, Sekot G, Bučková M, Godočíková J, Schäffer C, Farkašovský M, fort he catalase superfamily. (XLSX 22 kb) Obinger C, Polek B. Intracellular targeting of ascomycetous catalase- peroxidases (KatG1s). Arch Microbiology. 2013;195(6):393–402. 5. Li C, Shi L, Chen D, Ren A, Gao T, Zhao M. Functional analysis of the role of Abbreviations glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal CcP: Cytochrome c peroxidase; CldL: Chlorite dismutase-like protein; branching and the regulation of ganoderic acid biosynthesis in Ganoderma CTAB: Hexadecyltrimethylammonium ; HGT: Horizontal gene transfer; lucidum. Fungal Genet Biol. 2015;82:168–80. HMM: Hidden Markov model; KatG: Bifunctional catalase-peroxidase; 6. Narasaiah KV, Sashidhar RB, Subramanyam C. Biochemical analysis of LDS: Linoleate diol synthase; LiPOX: ; ML: Maximum oxidative stress in the production of aflatoxin and its precursor likelihood phylogeny; MnPOX: Manganese peroxidase; ORF: Open reading intermediates. Mycopathologia. 2006;162:179–89. frame; PAA: Peroxyacetic acid; PEG: Polyethylene glycol; PFL: Pyruvate 7. Nielsen KF, Gravesen S, Nielsen PA, Andersen B, Thrane U, Frisvad JC. formate-lyase; RT-qPCR: Quantitative real-time PCR; SOD: Superoxide Production of mycotoxins on artificially and naturally infested building dismutase; WSC: Cell-wall integrity & stress response component materials. Mycopathologia. 1999;145:43–56. Zámocký et al. BMC Genomics (2016) 17:763 Page 15 of 15

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