Metagenomic evidence for sulfur lithotrophy by Epsilonproteobacteria as the major energy source for primary productivity in a sub-aerial arctic glacial deposit, Borup Fiord Pass

Abstract

We combined free enenergy calculations and metagenomic analyses of an elemental sulfur (S(0)) deposit on the surface of Borup Fiord Pass Glacier in the Canadian High Arctic to investigate whether the energy available from different redox reactions in an environment predicts microbial metabolism. Many S, C, Fe, As, Mn, and [Formula: see text] oxidation reactions were predicted to be energetically feasible in the deposit, and aerobic oxidation of S(0) was the most abundant chemical energy source. Small subunit ribosomal RNA (SSU rRNA) gene sequence data showed that the dominant phylotypes were Sulfurovum and Sulfuricurvum, both Epsilonproteobacteria known to be capable of sulfur lithotrophy. Sulfur redox genes were abundant in the metagenome, but sox genes were significantly more abundant than reverse dsr (dissimilatory sulfite reductase)genes. Interestingly, there appeared to be habitable niches that were unoccupied at the depth of genome coverage obtained. Photosynthesis and [Formula: see text] oxidation should both be energetically favorable, but we found few or no functional genes for oxygenic or anoxygenic photosynthesis, or for [Formula: see text] oxidation by either oxygen (nitrification) or nitrite (anammox). The free energy, SSU rRNA gene and quantitative functional gene data are all consistent with the hypothesis that sulfur-based chemolithoautotrophy by Epsilonproteobacteria (Sulfurovum and Sulfuricurvum) is the main form of primary productivity at this site, instead of photosynthesis. This is despite the presence of 24-h sunlight, and the fact that photosynthesis is not known to be inhibited by any of the environmental conditions present. This is the first time that Sulfurovum and Sulfuricurvum have been shown to dominate a sub-aerial environment, rather than anoxic or sulfidic settings. We also found that Flavobacteria dominate the surface of the sulfur deposits. We hypothesize that this aerobic heterotroph uses enough oxygen to create a microoxic environment in the sulfur below, where the Epsilonproteobacteria can flourish.

Keywords: Epsilonproteobacteria; Sulfurovum; arctic; free energy; lithotrophy; metagenome; photosynthesis; sulfur.

FIGURE 1

Borup Fiord Pass Glacier. (A) Overview of the field site in 2009. (B) The spring source (site BF09-01) and sulfur varnish beside the spring (site BF09-02). (C) Sulfur deposit BF09-06 from which DNA was extracted for the metagenome.

FIGURE 2

Energy available from different redox reactions that could occur in the BF09-06 sulfur deposit. Ranges in the amount of energy reflect the range of uncertainty for substances that could not be detected (up to a maximum of the detection limit used for the assay). (A) The energy available per electron transferred. (B) The total energy available from the same reactions in the BF09-06 sulfur deposit, taking into account the total amount of each reactant present. The energy available from the aerobic oxidation of S⁰ is ringed in red.

FIGURE 3

SSU rRNA gene sequence data from the PCR amplicon library. (A) The SSU rRNA data at the phylum level, except that the proteobacteria have been split into classes. Phyla which are less than 0.01% in all samples are included in “other.” (B) Each OTU representing more than 5% of any sample, with all other OTUs included in “other.”

FIGURE 4

SSU rRNA gene sequence data from the metagenome shotgun library. Data are shown at the phylum level except that the proteobacteria have been split into classes. For some metagenome sequences the most closely related sequences in the Greengenes database were identified as proteobacteria but without specifying which Class. These are the sequences included in the “Proteobacteria (class not determined)” group. Phyla which represent less than 0.1% of sequences are included in “other.”

FIGURE 5

A maximum-likelihood tree representing the phylogenetic relationships of the dominant Epsilonproteobacteria OTUs in the BF09-06 sulfur deposit to Epsilonproteobacteria reported from other sites. Bootstrap values of less than 50 are not shown and these nodes are considered un-resolved. Thiomicrospira crunogena (Gammaproteobacteria) is the outgroup used to root the tree. Key: ^#environmental sequences from this study; *shorter sequence from 454 sequencing.

FIGURE 6

Normalized relative abundance (NRA) of functional genes in the metagenome.

FIGURE 7

Taxonomic origin of the best hits of metagenome genes which are found in high relative abundance produced by the MG-RAST analysis pipeline. Where a metagenome sequence has two or more equally good best hits the MG-RAST analysis retains all the best hit results. The “Epsilonproteobacteria/other” category is sequences for which there were two or more best hits against the same functional gene, but from different phyla. The taxonomic origin of these sequences cannot therefore be clearly determined. The “other” category includes all the other phyla represented within the best hits for each gene, and differ from gene to gene.

FIGURE 8

Reaction pathways which are likely to be significant in the BF09-06 sulfur deposit, based on the relative abundance of functional genes. Solid lines indicate oxidative reactions and dotted lines indicate reductive reactions. Blue lines indicate reactions that are catalyzed by enzymes whose genes are present in high relative abundance. The protein and gene names are written beside the relevant reaction. The black line is an abiotic reaction.