Draft Genome Sequences of Three Smithella spp. Obtained from a Methanogenic Alkane-Degrading Culture and Oil Field Produced Water.

Two draft genomes affiliated with Smithella spp. were obtained from a methanogenic alkane-degrading enrichment culture by single-cell sorting and metagenome contig binning, and a third was obtained by single-cell sorting of oil field produced water. Two genomes contained putative assABC genes encoding alkylsuccinate synthase, indicating genetic potential for fumarate activation of alkanes.

GENOME ANNOUNCEMENT

Smithella and Syntrophus (Syntrophaceae) have been implicated in long-chain n-alkane degradation by methanogenic communities (1, 2) but few have been cultivated (2) or sequenced. The sole draft Smithella genome (Smithella ME-1; NZ_AWGX00000000.1) was coassembled from single cells sorted from a methanogenic n-hexadecane-degrading culture (3), but the genome of the cultivated type strain, Smithella propionica LYP, has not been sequenced (4). A complete Syntrophus genome is available (S. aciditrophicus SB; NC_007759.1) but it harbors no known genes for anaerobic hydrocarbon biodegradation. Thus, additional genomes from these bacterial genera would contribute to understanding hydrocarbon bioremediation under anaerobic conditions.

Single-cell sorting (http://www.bigelow.org) of a methanogenic alkane-degrading culture (SCADC) (5) and produced water from an oil field in southern Alberta (6) yielded two single cells (SC-F21 and SC-D17, respectively) affiliated with Smithella. These were subjected to multiple displacement amplification and sequenced as single-cell amplified genomes (SAGs) using Illumina Mi-Seq (7), and then assembled de novo using CLC Genomics Workbench (CLC-Bio, USA) with a k-mer size of 40. A third draft genome was obtained by binning SCADC metagenome contigs (5) using sequence homology- and composition-based methods. All genomes were subjected to sequence decontamination (7) and annotated using RAST (8).

The SC-D17 draft genome is 1.6 Mbp on 271 scaffolds with 43% GC content, whereas SC-F21 is 1.6 Mbp on 245 scaffolds with 50% GC content. The SCADC draft genome is ~3.3 Mbp on 247 scaffolds (1,000–74,000 bp) with 44% GC content. Alignment and classification of the16S rRNA gene sequence (Silva Aligner) (http://www.arb-silva.de/aligner/) indicated >98% similarity to Smithella, supported by phylogenetic analysis placing SC-D17, SC-F21, and SCADC within the Smithella-affiliated clade (2). Therefore, the two SAGs were named Smithella sp. SC-D17 and Smithella sp. SC-F21, and the SCADC binned genome was named Smithella sp. SCADC. Two-way average nucleotide identity analysis between Smithella ME-1 (NZ_AWGX00000000.1) and the three new draft genomes (1000-bp window read size) revealed high similarity to Smithella SC-D17 (1524 fragments; 82% similarity) and Smithella SCADC (2842 fragments; 86% similarity) but lower pairwise similarity to Smithella SC-F21 (98 fragments; 85% similarity). Comparison of single-copy gene numbers in the draft genomes to Smithella aciditrophicus SB (NC_007759.1) indicates that the Smithella SC-D17 and SC-F21 genomes are partially (>70%) complete and Smithella SCADC is nearly (>95%) complete.

Sequence homologs of assA involved in alkane activation under sulfate- and nitrate-reducing conditions by Desulfatibacillum alkenivorans AK-01 and Azoarcus sp. HxN1, respectively (9, 10), were detected in Smithella spp. ME-1 (3, 11), SCADC (11), and SC-D17, but not SC-F21. In Smithella spp. SCADC and SC-D17, assA is present in gene clusters containing assB, assC, and masE homologs encoding alkylsuccinate synthase subunits (9, 10). The dsrAB and dsrMKJOP genes crucial for sulfate reduction were not detected in the three draft genomes, implying the inability to reduce sulfate, as in S. aciditrophicus SB (12). Whole-genome comparison is under way to study the functional roles of Smithella spp. in methanogenic alkane degradation.

Nucleotide sequence accession numbers.

The whole-genome shotgun projects for Smithella sp. SC-F21, Smithella sp. SC-D17, and Smithella sp. SCADC have been deposited at DDBJ/EMBL/GenBank under accession numbers JQIE00000000, JQOA00000000, and JQDQ00000000, respectively. The versions described in this paper are versions JQIE01000000, JQOA01000000, and JQDQ01000000.