Extremophiles (2016) 20:351–361 DOI 10.1007/s00792-016-0826-x ORIGINAL PAPER Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus) Konstantinos Papadimitriou2 · Panagiotis K. Baharidis2 · Anastasios Georgoulis1 · Marion Engel4,5 · Maria Louka1 · Georgia Karamolegkou1 · Aggeliki Tsoka1 · Jochen Blom7 · Bruno Pot8 · Piotr Malecki6 · Wojciech Rypniewski6 · Harald Huber3 · Michael Schloter4 · Constantinos Vorgias1 Received: 21 January 2016 / Accepted: 15 March 2016 / Published online: 25 March 2016 © Springer Japan 2016 Abstract Here we analyze the first complete genome metabolic activity of microbial consortia within hot sea sequence of Pyrococcus chitonophagus. The archaeon water ecosystems. Indeed, an obvious feature of the P. was previously suggested to belong to the Thermococ- chitonophagus genome is that it carries proteins show- cus rather than the Pyrococcus genus. Whole genome ing complementary activities for chitin degradation, i.e. phylogeny as well as whole proteome comparisons endo- and exo-chitinase, diacetylchitobiose deacetylase using all available complete genomes in Thermococca- and exo-β-D glucosaminidase activities. This finding les clearly showed that the species belongs to the Pyro- supports the hypothesis that compared to other Thermo- coccus genus. P. chitonophagus was originally isolated coccales species P. chitonophagus is adapted to chitin from a hydrothermal vent site and it has been described degradation. to effectively degrade chitin debris, and therefore is con- sidered to play a major role in the sea water ecology and Keywords Genome analysis · Pyrococcus chitonophagus · Chitinolytic activity · Hyperthermophilic archeon Communicated by H. Atomi. Electronic supplementary material The online version of this article (doi:10.1007/s00792-016-0826-x) contains supplementary material, which is available to authorized users. 5 Research Unit for Scientific Computing, Helmholtz Zentrum * Constantinos Vorgias München, Deutsches Forschungszentrum für Gesundheit und [email protected] Umwelt (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany 1 Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, 6 Institute of Bioorganic Chemistry, Polish Academy Panepistimiopolis Zografou, 15701 Athens, Greece of Sciences, Noskowskiego 12/14, Poznan 61‑704, Poland 2 Department of Food Science and Human Nutrition, 7 Bioinformatics and Systems Biology, Justus Liebig Agricultural University of Athens, Iera Odos 75, University, Giessen, Germany 11855 Athens, Greece 8 Institut Pasteur de Lille, U1019, UMR 8204, CIIL, Centre 3 Lehrstuhl für Mikrobiologie und Archäenzentrum Universität d’Infection et d’Immunité de Lille, Univ. Lille, CNRS, Regensburg, Universitätsstrasse 31, 93053 Regensburg, Inserm, CHU Lille, 59000 Lille, France Germany 4 Research Unit for Environmental Genomics, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany 1 3 352 Extremophiles (2016) 20:351–361 Abbreviations et al. 1995). In its original description, P. chitonophagus ORBs Origin recognition boxes was shown to grow chemoorganoheterotrophically by fer- HGT Horizontal gene transfer menting chitin as a sole carbon source or peptide mixtures CRISPRs Clustered regularly interspaced short pal- like yeast extract, meat extract, etc. (Huber et al. 1995). 0 indromic repeats It can also perform S respiration producing H2S. P. chi- Cas CRISPR-associated sequences tonophagus seems to be unable to utilize simple sugars GIs Genomic islands like glucose, N-acetylglucosamine, maltose or to degrade GlcNAc2 N,N’-Diacetylchitobiose complex polysaccharides like pectin, glycogen, agar, chi- GlcNAc N-Acetylglucosamine tosan, cellulose some of which can be catabolized by other Dac Diacetyl-chitobiose diacetylase pyrococci and thermococci. P. chitonophagus was misclas- GlmA Exo-β-D-glucosaminidase sified at the genus level and it has now been suggested to GlcN-GlcNAc N-Acetylchitobiose belong to the Pyrococcus rather than the Thermococcus Glk D -Glucosamine kinase genus (Lepage et al. 2004). Here we describe the analysis Pfk Phosphofructokinase of the first complete genome sequence of P. chitonophagus, GAP D -Glyceraldehyde 3-phosphate as well as a comparison with all available complete genome Fba Fructose-biphosphate aldolase sequences in Thermococcales. TpiA Triose-phosphate isomerase GAPOR Glyceraldehyde-3-phosphate-ferredoxin oxidoreducatse Materials and methods Pps Phosphoenolpyruvate synthase Pyk Pyruvate kinase Sequencing and assembly POR Pyruvate:ferredoxin oxidoreductase ACS Acetyl-CoA synthetase The library of P. chitonophagus was prepared using Nex- MBH Membrane-bound hydrogenase tera DNA Sample Preparation Kit (Illumina) following Nsr NADPH S0 reductase the manufacturer’s user guide. The initial concentra- tion of gDNA was measured using the Qubit® dsDNA HS Assay Kit (Life Technologies). The sample was then Introduction diluted accordingly to achieve the recommended DNA input of 50 ng at a concentration of 2.5 ng/µL. Subse- Thermococcales have attracted consistent attention from quently, the sample underwent the simultaneous fragmen- researchers because of their evolutionary significance as tation and addition of adapter sequences. These adapt- well as their biotechnological potential, linked to the pro- ers are utilized during a limited-cycle (5 cycles) PCR in duction of thermostable enzymes (Atomi 2005). Ther- which unique index was added to the sample. Follow- mococcales are strictly anaerobic and hyperthermophilic ing the library preparation, the final concentration of archaea belonging to the Euryarchaeota phylum. In this the library was measured (6.52 ng/µL) using the Qubit® order, three genera are distinguished: Pyrococcus (Fiala dsDNA HS Assay Kit (Life Technologies), and the aver- and Stetter 1986), Τhermococcus (Zillig et al. 1983) and age library size (1020 bp) was determined using the Agi- Palaeococcus (Takai et al. 2000). Most of these hyperther- lent 2100 Bioanalyzer (Agilent Technologies). The library mophilic archaea are organoheterotrophs that utilize pro- (12.5 pM) was sequenced using 600 Cycles v3 Reagent teins, starch and maltose with elemental sulfur (S0) or pro- Kit (Illumina) in MiSeq (Illumina). Genome was assem- tons as electron acceptors (Sapra et al. 2003). bled using NGEN (DNAstar) by Molecular Research LP The members of Pyrococcus, with higher optimum (MR DNA Shallowater, TX USA). growth temperature (95–103 °C) than Thermococcus (75– 93 °C) are ubiquitously present in natural high-temperature Annotation and comparative analysis of the P. environments, and are therefore considered to play a major chitonophagus genome role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. Complete genome The genome sequence of P. chitonophagus was annotated sequences are available for six Pyrococcus species (Cohen using the RAST server (Overbeek et al. 2014). Full chro- et al. 2003; Jun et al. 2011; Jung et al. 2012; Kawarabayasi mosome alignments were performed using Kodon (Applied et al. 1998; Lee et al. 2011; Robb et al. 2001). Maths) and ProgressiveMauve (Darling et al. 2010). Core Pyrococcus chitonophagus is a hyperthermophilic anaer- genome and singletons determination as well as whole obic archaeon, isolated from a deep sea hydrothermal vent genome phylogeny were performed with the EDGAR plat- site off the Mexican west coast at a depth of 2600 m (Huber form (Blom et al. 2009). 1 3 Extremophiles (2016) 20:351–361 353 Fig. 1 Genome map of P. chitonophagus. Tracks from the periphery of the map towards the center: CDSs in the forward strand (red), CDSs in the reverse strand (blue), GC plot, GC skew Additional analysis 2,153 protein coding genes covering more than 90 % of the genome sequence. The genome also carries 48 tRNA The map of the P. chitonophagus genome was constructed and 4 rRNA genes (2 copies of 5S rRNA, 1 copy of 16S using DNAPlotter software (Carver et al. 2009). The OriC rRNA and 1 copy of 23S rRNA genes). A single OriC was of P. chitonophagus was predicted using the Ori-Finder 2 identified using the Ori-Finder 2 tool at regions spanning tool (Luo et al. 2014). Clustered regularly interspaced short positions 247,894 and 248,700, right upstream of the cdc6 palindromic repeats (CRISPRs) were analyzed within the and DNA pol II genes. Manual investigation indicated the CRISPRcompar web-service (Grissa et al. 2008). When- presence of two origin recognition boxes (ORBs) inverted ever necessary, the BLAST suite was used for sequence repeats showing high similarity to ORBs described previ- similarity searches (Altschul et al. 1990). Functional ously for Pyrococcus abyssi (Matsunaga et al. 2003) (Sup- annotation was performed using the eggNOG database plementary Fig. 1). (Powell et al. 2012). Metabolic networks of the archaeon were automatically constructed using the KAAS (Moriya Comparative and evolutionary genomics et al. 2007). The pathways presented in this study were also manually curated. PanFunPro was employed for pan- Thermococcus chitonophagus was suggested to belong genome analysis (Lukjancenko et al. 2013). to the Pyrococcus rather than the Thermococcus genus (Lepage et al. 2004). To further investigate this matter we performed full chromosome alignments using all avail- Results and discussion able complete genome sequences in Thermococcales. The genome did not align with Thermococcus