Complete Genome Sequence of Bacteroidales Strain CF from a Chloroform-Dechlorinating Enrichment Culture.

Bacteroidales strain CF is the most abundant nondechlorinating organism in a Dehalobacter-containing enrichment culture that consistently reductively dechlorinates >50 mg/liter chloroform or 1,1,1-trichloroethane (methyl chloroform). We assembled and closed the complete genome sequence of this organism from the metagenomic sequencing data for enrichment cultures. This organism is predicted to ferment l-lactate and ethanol.

GENOME ANNOUNCEMENT

ACT-3 is an enrichment culture used for bioaugmentation to detoxify groundwater contaminated with 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane (1,1-DCA), or chloroform (CF) (1). It is grown in prereduced defined mineral medium amended with ~50 mg/liter 1,1,1-TCA as an electron acceptor and a mixture of methanol, ethanol, and lactate as electron donors. The ACT-3 culture is dominated by two Dehalobacter strains that make up ~70% of the community. In this culture, 1,1,1-TCA is reductively dechlorinated via 1,1-DCA to monochloroethane and CF is reductively dechlorinated to dichloromethane (2). A subculture of the ACT-3 culture is maintained identically except that 1,1,1-TCA is replaced by CF. This subculture is dominated (~80%) by only one of the two Dehalobacter strains from the parent culture (3). The parent and CF subcultures also share a nondechlorinating organism that is the next most abundant (8 to 9%) in both cultures (3). We have named this organism Bacteroidales strain CF based on 16S rRNA phylogeny. This organism is of interest because it ferments in the presence of concentrations of CF or 1,1,1-TCA that usually inhibit microbial processes, including methanogenesis (4, 5) and reductive dechlorination (6, 7).

The complete genome sequence of Bacteroidales strain CF was assembled from the metagenomes of ACT-3 and the CF subculture. ACT-3 was sequenced using 454 pyrosequencing, including paired-end sequencing of an 8-kb insert library, and the sequence was assembled with Newbler version 2.5 (8). The CF subculture was sequenced using paired-end Illumina technology, and the sequence was assembled with AbySS version 1.3.4 (9). Previously, we reported the assembly of two complete Dehalobacter genomes from these two metagenomes (3). The contigs from the subculture metagenome that mapped to either of the two Dehalobacter genomes were removed to obtain the contigs of microbes other than Dehalobacter spp. These contigs were compared to scaffolds generated from the ACT-3 metagenome. A circular scaffold comprising twenty non-Dehalobacter contigs was constructed. Nineteen gaps were resolved in silico with a previously described method (3). The last gap, a highly repetitive region, was resolved by long reads generated from additional Sanger sequencing guided by PCR. Having two metagenome data sets enabled the complete assembly: the ACT-3 metagenome provided long-distance mate-pair constraints for scaffolding despite having insufficient coverage (~14×) to close the genome, while the CF metagenome provided sufficient coverage (~40×).

The genome of Bacteroidales strain CF is ~2.66 Mb, with an average G+C content of 42.7%. It contains 9 rRNA genes (3 16S rRNA genes), 37 tRNA genes, and ~2,310 coding sequences. It was annotated with RAST (10) and BASys (11). The genes required for the fermentation of l-lactate, ethanol, and glucose were identified. Genes encoding various hydrogenases and an incomplete citric acid cycle were also found. No essential genes involved in the Wood-Ljungdahl pathway were identified. The closest genome sequence is a draft genome sequence of Alistipes putredinis DSM 17216 (GenBank accession no. ABFK00000000), whose 16S rRNA sequences share only ~86% identity with those of Bacteroidales strain CF.

Nucleotide sequence accession number.

This genome sequence has been deposited in GenBank with the accession no. CP006772.