ABSTRACT

The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia.

ANNOUNCEMENT

Naganishia liquefaciens (phylum Basidiomycota, class Tremellomycetes) strain N6 was isolated from the Japan Trench (6,500 m) (1, 2). Sediment samples were spread onto yeast extract-peptone-dextrose (YPD) plates (3) containing penicillin and streptomycin sulfate, and colonies were isolated. Although initially named Cryptococcus liquefaciens based on a comparison of the 18S rDNA with that of Cryptococcus albidus (1, 2), Cryptococcus albidus was subsequently assigned to the genus Naganishia; Cryptococcus liquefaciens was therefore renamed Naganishia liquefaciens (4, 5). Strain N6 is tolerant to heavy metals (1, 2, 6). Many deep-sea microorganisms have evolved to survive under extreme conditions, and their characterization is potentially crucial for the production of useful biomolecules.
Strain N6 was cultivated on YPD medium (3) at 30°C, and genomic DNA was prepared (Dr. GenTLE kit, TaKaRa Bio). Whole-genome sequencing was performed using the Illumina MiSeq platform. Three paired-end (kit, TruSeq; 34,806,922 reads; read length, 150 or 300 bp; insert sizes, 400 to 550 bp) and three mate pair (kit, Nextera mate pair; 25,211,788 reads; read length, 250 bp; insert sizes, 4,000 to 10,000 bp) libraries were generated (total, 14.7 Gbp). Default parameters were used except where otherwise noted. The reads were trimmed using Platanus_trim v1.0.2 (http://platanus.bio.titech.ac.jp/pltanus_trim). De novo assembly was performed by Platanus v1.2.1 (7), inputting all but the 10-kbp mate pair libraries. Misassemblies were corrected based on the physical coverage of the 10-kbp mate pairs. Some gaps were filled by additional Sanger sequencing of the PCR products of gap-flanking primers (44 reads; SRA accession number DRR244395; alignment tool, BLASTN). The complete mitochondrial genome (GenBank accession number BLZA01000059.1) was constructed using Platanus (“assemble -n 200” command). The numbers of resulting scaffolds and contigs, the total length, the scaffold N50 value, the contig N50 value, the gap rate, and the GC content were 59, 87, 19.44 Mbp, 1.03 Mbp, 0.62 Mbp, 0.10%, and 53.38%, respectively.
To determine strain N6’s gene structure, total RNA from cells grown in YPD plus adenine (YPAD) or YPAD containing 10 mM CuSO4 was prepared (Nucleospin RNA kit, Macherey‐Nagel). Four transcriptome sequencing (RNA-seq) libraries (two replicates for each condition) were prepared [TruSeq kit with poly(A) selection], and RNA-seq was performed using an Illumina MiSeq instrument (read length, 300 bp), resulting in 37,287,236 reads (total 8.10 Gbp). The protein-coding gene structure on the scaffolds was predicted using FunGAP v1.0.0 (8) and proteins from Cryptococcus neoformans and Cryptococcus gattii (GenBank accession numbers GCA_000091045.1 and GCA_000185945.1, respectively). A total of 6,883 genes were predicted. The average lengths of transcripts and coding DNA sequences (CDS) were 1,999 bp and 1,621 bp, respectively. In addition, 38,748 introns were identified, with 93.7% of genes containing at least one intron.
We downloaded 113 genomes in the class Tremellomycetes and the genome of Ustilago maydis JCM 2005 as an outgroup from the GenBank database. Single-copy orthologs were identified using BUSCO v4.0.6 (9) with the basidiomycota_odb10 data set (1,764 orthologs). In the strain N6 genome, 1,613 (91.3%) benchmarking universal single-copy ortholog (BUSCO) complete genes were detected. For each ortholog group, protein sequences were aligned using MAFFT v7.455 (10), and gaps were removed. From the concatenated alignment, a maximum-likelihood tree was reconstructed using IQ-TREE v1.6.12 (11) with the LG+I+G4+F model and 1,000 bootstrap replicates. The resulting tree (Fig. 1) suggests that strain N6 belongs to the genus Naganishia.
FIG 1
FIG 1 Phylogenetic tree of genomes in the class Tremellomycetes. N. liquefaciens strain N6 is shown in red. Ustilago maydis JCM 2005 is used as an outgroup. The number of bootstrap replicates is 1,000, and the percentages of bootstrap supports (1% to 100%) are shown as the numbers near the nodes (only the bootstrap supports of <100 are displayed). Branch lengths are based on the number of substitutions per site, as indicated by the scale bar at the bottom. The number of sites in the multiple alignment is 133,605 (amino acids).

Data availability.

The raw reads and the draft genome have been deposited in DDBJ/ENA/GenBank under BioProject accession number PRJDB10172 and the whole-genome shotgun project number BLZA00000000.1, respectively. The SRA accession numbers for paired-end reads are DRR237062, DRR237063, and DRR237064. The SRA accession numbers for mate pair reads are DRR237065, DRR237066, and DRR237067. The SRA accession numbers for RNA-seq reads are DRR237068, DRR237069, DRR237070, and DRR237071. The SRA accession number for Sanger reads is DRR244395.

ACKNOWLEDGMENT

This study was supported partly by Grants-in-Aid for Scientific Research on Innovative Areas (15H05979 to T.I. and 15H05974 to H.I.), for Scientific Research (A) (18H03985 to H.I.), and for Scientific Research (B) (19H03206 to T.I. and 18H02371 to H.T.) from the Japan Society for the Promotion of Science (JSPS).

REFERENCES

1.
Miura T, Abe F, Inoue A, Usami R, Horikoshi K. 2001. Purification and characterization of novel extracellular endopolygalacturonases from a deep-sea yeast, Cryptococcus sp. N6, isolated from the Japan Trench. Biotechnol Lett 23:1735–1739.
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Abe F, Miura T, Nagahama T, Inoue A, Usami R, Horikoshi K. 2001. Isolation of a highly copper-tolerant yeast, Cryptococcus sp. N6, from the Japan Trench and the induction of superoxide dismutase activity by Cu2+. Biotechnol Lett 23:2027–2034.
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Sherman F. 2002. Getting started with yeast. Methods Enzymol 350:3–41.
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Liu X-Z, Wang Q-M, Göker M, Groenewald M, Kachalkin AV, Lumbsch HT, Millanes AM, Wedin M, Yurkov AM, Boekhout T, Bai F-Y. 2015. Towards an integrated phylogenetic classification of the Tremellomycetes. Stud Mycol 81:85–147.
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Teh A-H, Kanamasa S, Kajiwara S, Kumasaka T. 2008. Structure of Cu/Zu superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6. Biochem Biophys Res Commun 374:475–478.
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Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T. 2014. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res 24:1384–1395.
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Min B, Grigoriev IV, Choi I-G. 2017. FunGAP: Fungal Genome Annotation Pipeline using evidence-based gene model evaluation. Bioinformatics 33:2936–2937.
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Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31:3210–3212.
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Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 9Number 4719 November 2020
eLocator: 10.1128/mra.00827-20
Editor: Antonis Rokas, Vanderbilt University

History

Received: 16 July 2020
Accepted: 27 October 2020
Published online: 19 November 2020

Contributors

Authors

Yong-Woon Han
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Present address: Yong-Woon Han, Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan; Yumiko Kurokawa, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan.
Rei Kajitani
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Hiroya Morimoto
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Maierdan Palihati
Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
Yumiko Kurokawa
Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
Present address: Yong-Woon Han, Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan; Yumiko Kurokawa, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan.
Rie Ryusui
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Bilge Argunhan
Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
Hideo Tsubouchi
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
Fumiyoshi Abe
College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Kanagawa, Japan
Susumu Kajiwara
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Hiroshi Iwasaki
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan

Editor

Antonis Rokas
Editor
Vanderbilt University

Notes

Address correspondence to Takehiko Itoh, [email protected].
Yong-Woon Han and Rei Kajitani contributed equally to this work. Author order was determined by alphabetical order of last name.

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