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Draft Genome Sequence of Pseudomonas sp. Strain CCA1, Isolated from Leaf Soil Hironaga Akita,a Zen-ichiro Kimura,b Tamotsu Hoshinoa,c Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Kagamiyama, Higashi-Hiroshima, Hiroshima, Japana; Department of Civil and Environmental Engineering, National Institute of Technology, Kure College, Aga, Minami, Kure, Hiroshima, Japanb; Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo, Japanc
Pseudomonas sp. strain CCA1 was isolated from leaf soil collected in Higashi-Hiroshima City in Hiroshima Prefecture, Japan. Here, we present a draft genome sequence of this strain. The genome consists of 24 contigs for a total of 6,993,992 bp, 8,917 predicted coding sequences, and a GC content of 67.2%. Received 13 October 2016 Accepted 14 October 2016 Published 8 December 2016 Citation Akita H, Kimura Z-I, Hoshino T. 2016. Draft genome sequence of Pseudomonas sp. strain CCA1, isolated from leaf soil. Genome Announc 4(6):e01371-16. doi:10.1128/ genomeA.01371-16. Copyright © 2016 Akita et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Hironaga Akita,
[email protected].
L
ignin is an aromatic polymer and the second most abundant organic polymer on Earth (1). Thus, effective utilization of lignin could support second-generation biofuel production from lignocellulosic biomass (2). Phanerochaete chrysosporium, Rhodococcus erythropolis, and Streptomyces coelicolor are able to assimilate lignin as a carbon source (3). However, methods for their culture are intricate and their growth is relatively slow, making them unsuitable for industrial production of second-generation biofuels. We therefore screened for lignin-degrading bacteria with rapid growth rates and high capacities for lignin degradation. This led to the isolation of Pseudomonas sp. strain CCA1 (strain number HUT-8136) from leaf soil (4). After Pseudomonas sp. strain CCA1 was aerobically cultured overnight at 37°C in nutrient broth (Kyokuto), the genomic DNA was extracted and purified using an illustra bacteria genomicPrep mini spin kit (GE Healthcare) according to the manufacturer’s instructions. The purity and concentration of the genomic DNA and double-stranded DNA were measured using a NanoDrop (Thermo Scientific) and a Quant-iT dsDNA BR Assay Kit (Invitrogen), respectively. After fragmenting the genomic DNA (7.8 g) into approximately 20-kb pieces using a g-TUBE (Covaris), the resultant fragments were ligated to SMRTbell sequencing adapters using an SMRTbell Template Prep Kit 1.0 (Pacific Biosciences), which yielded the SMRTbell libraries. The library size was measured using an Agilent 2200 TapeStation (Agilent Technologies). The SMRTbell libraries were then bound to polymerases and sequencing primers using a DNA/Polymerase Binding Kit P6 v2 (Pacific Biosciences), which yielded the sequencing templates. After the concentration of the sequencing templates was calculated using Binding Calculator Version 2.3.1.1 (Pacific Biosciences), the templates were bound to MagBeads using a MagBead Kit (Pacific Biosciences) and loaded onto singlemolecule real-time (SMRT) cells 8 Pac V3 (Pacific Biosciences). The sequencing was performed using the PacBio RS II platform (Pacific Biosciences). The raw data included 63,138 reads at 125-fold coverage and were assembled de novo, using SMRT Analysis v2.3.0 (Pacific Biosciences) (5) to filter the subreads. The genome sequence consisted of 6,993,992 bp with a GC content of 67.2%. The assembly generated 24 contigs with an N50 contig size of 6,935,186 bp. Genome
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annotation was performed using CRITICA (6) and Glimmer2 (7), and 8,917 predicted coding sequences were identified. In addition, 68 tRNA genes and 17 rRNA genes were identified using tRNAScan-SE (8) and BLASTN (9), respectively. Accession number(s). The nucleotide sequence and annotation data for the Pseudomonas sp. strain CCA1 draft genome have been deposited in DDBJ/EMBL/GenBank under accession numbers BDGS01000001 to BDGS01000024. ACKNOWLEDGMENT We are grateful to all members of the Bio-Conversion Research Group at the Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Sciences and Technology (AIST), for their technical assistance and valuable discussion.
FUNDING INFORMATION This work was supported in part by the Science and Technology Research Partnership for Sustainable Development (SATREPS), under the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA).
REFERENCES 1. Boerjan W, Ralph J, Baucher M. 2003. Lignin biosynthesis. Annu Rev Plant Biol 54:519 –546. http://dx.doi.org/10.1146/annurev.arplant.54.031902.134938. 2. Islam ZU, Zhisheng Y, Hassan El B, Dongdong C, Hongxun Z. 2015. Microbial conversion of pyrolytic products to biofuels: a novel and sustainable approach toward second-generation biofuels. J Ind Microbiol Biotechnol 42: 1557–1579s. http://dx.doi.org/10.1007/s10295-015-1687-5. 3. Ahmad M, Taylor CR, Pink D, Burton K, Eastwood D, Bending GD, Bugg TD. 2010. Development of novel assays for lignin degradation: comparative analysis of bacterial and fungal lignin degraders. Mol Biosyst 6:815– 821. http://dx.doi.org/10.1039/b908966g. 4. Akita H, Kimura Z, Yusoff MZ, Hoshino T. 2016. Isolation of Pseudomonas sp. strain CCA1 from leaf soil and preliminary characterization its ligninolytic activity. JSM Biotechnol Bioeng 3:1062. https://www .jscimedcentral.com/Biotechnology/biotechnology-3-1062.pdf. 5. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. http://dx.doi.org/ 10.1038/nmeth.2474.
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Akita et al.
6. Badger JH, Olsen GJ. 1999. CRITICA: coding region identification tool invoking comparative analysis. Mol Biol Evol 16:512–524. http:// dx.doi.org/10.1093/oxfordjournals.molbev.a026133. 7. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: 4636 – 4641. http://dx.doi.org/10.1093/nar/27.23.4636.
2 genomea.asm.org
8. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964. http://dx.doi.org/10.1093/nar/25.5.0955. 9. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403– 410. http://dx.doi.org/10.1016/ S0022-2836(05)80360-2.
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