Reconstruction of a Nearly Complete Pseudomonas Draft Genome Sequence from a Coalbed Methane-Produced Water Metagenome

The draft genome sequence of Pseudomonas stutzeri strain K35 was separated from a metagenome derived from a produced water microbial community of a coalbed methane well. The genome encodes a complete nitrogen fixation pathway and the upper and lower naphthalene degradation pathways.

tracted in silico from the metagenome of a produced water sample, which was taken from the Pocahontas 3 coal seam in the Central Appalachian Basin at a depth of 1,912 ft. Genomic DNA was isolated using the PowerSoil DNA kit (MoBio Laboratories, Inc., Carlsbad, CA). A paired-end (2 ϫ 250) library was prepared using the Nextera XT kit and the MiSeq reagent kit v2 (Illumina, Inc., San Diego, CA) and run with 500 cycles. The resultant 430,878 reads were assembled with metaSPAdes (version 3.7.0) (1). The metagenome assembly yielded 12,264 total contigs (12,332,388 bp), with an N 50 of 4,275 and a GC% of 56.52 (SRA accession no. SRP079849). The metagenome was binned with VizBin (2) and 22.5% of the contigs were assigned to the genus Pseudomonas. The metagenome reads were mapped to the contigs of the Pseudomonas genome bin using CLC genomics workbench (Qiagen) and mapped reads were reassembled with SPAdes (default parameters; contigs added as trusted contigs). The final contigs were extended using paired-read iterative contig extension (PRICE) (3).
The draft genome bin contained seven contigs (139,857 bp) that mapped to other Pseudomonas plasmids (BLASTn best hit) and may be part of a genomic island (GI) within the chromosome or an extrachromosomal plasmid. The GI/plasmid encodes for numerous type IV secretion system proteins, DNA replication machinery (e.g., DNA helicase, DNA topoisomerase), and mobilization elements (e.g., transposases and integrases).
The genome was annotated with Rapid Annotations using Subsystem Technology (RAST) version 2.0, using the RASTtk pipeline (7,8), and the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/). Initial analysis revealed the presence of complete nitrogen fixation and denitrification pathways. The upper and lower naphthalene degradation pathways were also found. The genome encodes for alginate biosynthesis pathways and a number of genes involved in oxygen stress (catalases, peroxidases, and superoxide dismutases). This genome provides a platform for understanding the role Pseudomonas spp. play in the conversion of coal to methane and the ecology of coalbed methane systems.
Accession number(s). This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. MCAL00000000. The version described in this paper is version MCAL01000000.

ACKNOWLEDGMENTS
This technical effort was performed in support of the National Energy Technology Laboratory's ongoing research under the RES contract DE-FE0004000.
This project was funded by the Department of Energy, National Energy Technology Laboratory, an agency of the United States Government, through a support contract with AECOM. Neither the United States Government nor any agency thereof, nor any of their employees, nor AECOM, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.