Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia, phylum Chloroflexi

Abstract

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated 'DEH-J10' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO2 or oxidation of organics completely to CO2. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.

Figure 1

Phylogenetic tree based on 16S rRNA genes to examine the phylogeny of the single cell DEH-J10 (highlighted) compared with cultivated and uncultivated members of the Dehalococcoidia and other major groups of the Chloroflexi. The tree is based on the neighbor-joining algorithm with bootstrap resampling (1000 times). Nodes with bootstrap values ⩾50% are indicated by filled circles (●) and nodes with bootstrap values of ⩾90% are indicated by open circles (○). Deinococcus frigens (GenBank no. AJ585982) was used as an outgroup to root the tree. The scale bar represents 10% sequence divergence. Previously defined Chloroflexi subphylums ‘II' and ‘IV' (Inagaki et al, 2006) are also indicated for comparative purposes.

Figure 2

Quantification of ‘total' bacterial 16S rRNA genes and genes amplified by specific primers designed for the 16S rRNA gene of the single cell DEH-J10 through depths of the Aarhus Bay sediment core. ‘Total' Bacteria are represented by filled circles (●), DEH-J10 by open circles (○).

Figure 3

Schematic depiction of the overall metabolic and phenotypic features of single cell DEH-J10, as predicted from single-cell genome sequencing and gene annotations. BCAA,  branched chain amino acids; ETF, electron transfer protein complex; HDR, heterodisulfide reductase-like proteins; mvhD, methyl-violgen-reducing hydrogenase delta subunit; DMSO, dimethyl sulfoxide; DMS, dimethyl sulphide. X–unknown electron carrier.