Draft Genome Sequence of Uncultivated Firmicutes (Peptococcaceae SCADC) Single Cells Sorted from Methanogenic Alkane-Degrading Cultures

Abstract

The draft genome of an uncultivated bacterium affiliated with the Peptococcaceae was reconstructed by co-assembling Illumina MiSeq sequences from three single cells sorted by microfluidics from two methanogenic alkane-degrading cultures. Peptococcaceae SCADC (short-chain alkane-degrading culture) may be genetically capable of anaerobic alkane activation by fumarate addition in the absence of sulfate.

GENOME ANNOUNCEMENT

We previously described a methanogenic SCADC (short-chain alkane-degrading culture) enriched from an oil sands tailings pond (1). During incubation at ~25° C and several passages under strictly methanogenic conditions for >2 years, SCADC produced methane with stoichiometric depletion of n-hexane, -heptane, -octane, -decane, 2-methylpentane, and methylcyclopentane (1). Subsamples of SCADC and a methanogenic iso-alkane-degrading culture (ISODC; N. Abu Laban, A. Dao, K. Semple, J. Foght, submitted for publication), incubated under parallel conditions, were subjected to microfluidic single-cell sorting (https://www.bigelow.org). Two cells from SCADC and one from ISODC were subjected to multiple displacement amplification, sequenced as single-amplified genomes (SAGs) using Illumina MiSeq (http://tagc.med.ualberta.ca/), and individually assembled de novo using SPAdes version 3.1 (2) with a read correction module and k-mer sizes of 21, 33, 45, 55, 77, 99, and 127. De novo assembly for each sorted cell produced 200 to 206 contigs of 1,000-bp minimum length. Two-way nucleotide identity analysis (http://enve-omics.ce.gatech.edu/ani) of the three genomes yielded an average of 4,136 fragments (500-bp window read size) having 99.24% mean identity and sharing identical 16S rRNA genes. Based on this similarity, all Illumina reads from the three cells were co-assembled de novo using SPAdes (2), as above.

To exclude contaminating sequences, scaffolds were fragmented in silico to 500 bp followed by BLASTx searches against the NCBI NR database. Fragments were assigned to taxa using MEGAN version 5.0 (3) with a minimum bit-score support of 100. Contigs with >50% of their fragments having hits to Firmicutes sequences were retained; the remainder were defined as having weak taxon support. All contigs were then subjected to tetranucleotide frequency analysis followed by clustering (4) in R (http://www.r-project.org). Contigs with weak taxon support that did not cluster with the Firmicutes-affiliated contigs were removed from the final genomic bin. The draft genome was named Peptococcaceae SCADC (i.e., identified at the family level) because phylogenetic and BLASTn analysis of the 16S rRNA gene sequence showed ≤90% identity to Desulfotomaculum and Pelotomaculum (the closest cultivated matches), precluding classification to the genus level.

The Peptococcaceae SCADC draft genome is ~2.6 Mbp contained in 259 scaffolds with an N₅₀ of 23,229 bp and an average 42.9% GC content. Annotation using NCBI Prokaryotic Genome Annotation Pipeline predicted 2,208 coding sequences. Ninety-five single-copy genes were recovered using the gene list compiled by Albertsen et al. (5) versus 108 and 109 genes detected in Pelotomaculum thermopropionicum SI (NC_009454.1) and Desulfotomaculum acetoxidans DSM 771 (NC_013216.1), suggesting that the draft genome is >87% complete.

Genome annotation and phylogenetic analysis detected a putative gene encoding AssA, 47% similar to the AssA subunit in Desulfoglaeba alkanexedens ALDC (ADJ51097). Therefore, Peptococcaceae SCADC also may be capable of alkane addition to fumarate during anaerobic hydrocarbon degradation. However, whereas Desulfotomaculum spp. harbor dsrAB genes and reduce sulfate and sulfite (6), the Peptococcaceae SCADC draft genome lacks obvious dsrAB and dsrMKJOP orthologs. Therefore, like Pelotomaculum (7), it likely is incapable of complete sulfate reduction. Genomic and metatranscriptomic analyses of Peptococcaceae SCADC in alkane-degrading enrichment cultures (1) is under way to study its potential role in alkane degradation under methanogenic conditions.

Nucleotide accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number JJNX00000000. The draft genome described in this paper is version JJNX02000000.