Comparative Genomic Analysis of <i>Sulfurospirillum cavolei</i> MES Reconstructed from the Metagenome of an Electrosynthetic Microbiome

<div><p><i>Sulfurospirillum</i> spp. play an important role in sulfur and nitrogen cycling, and contain metabolic versatility that enables reduction of a wide range of electron acceptors, including thiosulfate, tetrathionate, polysulfide, nitrate, and nitrite. Here we describe the assembly of a <i>Sulfurospirillum</i> genome obtained from the metagenome of an electrosynthetic microbiome. The ubiquity and persistence of this organism in microbial electrosynthesis systems suggest it plays an important role in reactor stability and performance. Understanding why this organism is present and elucidating its genetic repertoire provide a genomic and ecological foundation for future studies where <i>Sulfurospirillum</i> are found, especially in electrode-associated communities. Metabolic comparisons and in-depth analysis of unique genes revealed potential ecological niche-specific capabilities within the <i>Sulfurospirillum</i> genus. The functional similarities common to all genomes, <i>i</i>.<i>e</i>., core genome, and unique gene clusters found only in a single genome were identified. Based upon 16S rRNA gene phylogenetic analysis and average nucleotide identity, the <i>Sulfurospirillum</i> draft genome was found to be most closely related to <i>Sulfurospirillum cavolei</i>. Characterization of the draft genome described herein provides pathway-specific details of the metabolic significance of the newly described <i>Sulfurospirillum cavolei</i> MES and, importantly, yields insight to the ecology of the genus as a whole. Comparison of eleven sequenced <i>Sulfurospirillum</i> genomes revealed a total of 6246 gene clusters in the pan-genome. Of the total gene clusters, 18.5% were shared among all eleven genomes and 50% were unique to a single genome. While most <i>Sulfurospirillum</i> spp. reduce nitrate to ammonium, five of the eleven <i>Sulfurospirillum</i> strains encode for a nitrous oxide reductase (<i>nos</i>) cluster with an atypical nitrous-oxide reductase, suggesting a utility for this genus in reduction of the nitrous oxide, and as a potential sink for this potent greenhouse gas.</p></div

Phylogenetic tree of <i>Sulfurospirillum</i>.

<p>The unrooted tree was constructed using the Neighbor-Joining method with near complete 16S rRNA gene sequences, with a bootstrap value of 1000. Distance bar represents one substitution per 100 nucleotide positions. Strains with sequenced genomes (draft or complete) are denoted in bold. <i>S</i>. <i>cavolei</i> MES is highlighted in blue. The accession numbers for the 16S rRNA gene from <i>S</i>. <i>cavolei</i> NBRC were not publicly available and were alternatively identified by BLASTn analysis of the <i>S</i>. <i>cavolei</i> genome using the 16S rRNA gene from <i>S</i>. <i>cavolei</i> MES as query.</p

Reconstruction of central metabolism of <i>Sulfurospirillum</i> species.

<p>All enzymes or enzyme complexes depicted are found in all eleven sequenced genomes. Enzymes denoted with a red border are unique to a single genome or found in a subset of genomes. DNRA, dissimilatory nitrate reduction to ammonium; Nap, nitrate reductase; Nrf, nitrite reductase; Nif, nitrogen fixation; Nos, nitrous oxide reductase; Nor, nitric oxide reductase; cyt. <i>c</i> oxidase, cytochrome <i>c</i> oxidase; NADH-I, NADH-quinone oxidoreductase; ε-NADH I, ferredoxin/flavodoxin-quinone oxidoreductase; TTR, tetrathionate reductase; PSR, polysulfide reductase; Mcc, respiratory sulfite reductase Q, quinone; MK, menaquinone.</p

Neighbor-joining tree of <i>S</i>. <i>cavolei</i> MES iron hydrogenase.

<p>Tree consists of amino acid sequences closely related to <i>Sulfurospirillum cavolei</i> MES [FeFe] hydrogenase large subunit (denoted in blue), with a bootstrap value of 1000.</p

Operon organization of nitrous oxide reduction pathway.

<p>Nitrous oxide reductase gene cluster of <i>Wolinella succinogens</i> (top) and <i>Sulfurospirillum</i> spp. (bottom). Color scheme was maintained from reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151214#pone.0151214.ref018" target="_blank">18</a>]. Green ORFs encode for nitrous oxide reductase, yellow ORFs encode a conserved trans-membrane protein, blue ORFs encode for <i>nos</i> accessory proteins, purple ORFs encode for 4Fe-4S proteins, red ORFs encode for <i>c-</i>type cytochromes, and white ORFs (labeled 1–5) represent additional predicted genes not found in the <i>W</i>. <i>succinogens nos</i> cluster. Putative function of additional ORFs: 1. Hypothetical protein (Locus tag: OA34_06690), 2. ABC transporter permease (OA34_10365), 3. ABC transporter ATP-binding protein (OA34_10360), 4. Hypothetical protein (OA34_10355), 5. Hypothetical protein (OA34_06670). Note: ORFs not drawn to scale, the length of the predicted protein (in amino acid residues) encoded by each gene is provided.</p

Neighbor-joining tree of the NosZ amino acid sequence.

<p>Bootstrap value of 1000. <i>S</i>. <i>cavolei</i> MES is denoted in blue.</p

Parsimony pan-genome tree for eleven <i>Sulfurospirillum</i> proteomes.

<p>Phylogeny is based upon the gene family content (presence or absence of homologous genes) of each proteome, and the parsimony pan-genome tree was constructed with a midpoint root. <i>S</i>. <i>cavolei</i> MES is denoted in blue. Inset: Next to each organism is the average nucleotide identity (two way ANI) and average amino acid identity (AAI) compared to <i>S</i>. <i>cavolei</i> MES.</p

Pan-genome analysis of 11 <i>Sulfurospirillum</i> genomes.

<p>Pan-genome matrix partitioned into core, soft core, shell, and cloud components (core = all 11 species, soft core = ≥ 10, shell = 3–9, and cloud = ≤ 2).</p

Eco-physiological characteristics of members from genus <i>Sulfurospirillum</i>.

<p>Eco-physiological characteristics of members from genus <i>Sulfurospirillum</i>.</p

Long-term Operation of Microbial Electrosynthesis Systems Improves Acetate Production by Autotrophic Microbiomes

Microbial electrosynthesis is the biocathode-driven production of chemicals from CO₂ and has the promise to be a sustainable, carbon-consuming technology. To date, microbial electrosynthesis of acetate, the first step in order to generate liquid fuels from CO₂, has been characterized by low rates and yields. To improve performance, a previously established acetogenic biocathode was operated in semi-batch mode at a poised potential of −590 mV vs SHE for over 150 days beyond its initial development. Rates of acetate production reached a maximum of 17.25 mM day^–1 (1.04 g L^–1 d^–1) with accumulation to 175 mM (10.5 g L^–1) over 20 days. Hydrogen was also produced at high rates by the biocathode, reaching 100 mM d^–1 (0.2 g L^–1 d^–1) and a total accumulation of 1164 mM (2.4 g L^–1) over 20 days. Phylogenetic analysis of the active electrosynthetic microbiome revealed a similar community structure to what was observed during an earlier stage of development of the electroacetogenic microbiome. <i>Acetobacterium</i> spp. dominated the active microbial population on the cathodes. Also prevalent were <i>Sulfurospirillum</i> spp. and an unclassified Rhodobacteraceae. Taken together, these results demonstrate the stability, resilience, and improved performance of electrosynthetic biocathodes following long-term operation. Furthermore, sustained product formation at faster rates by a carbon-capturing microbiome is a key milestone addressed in this study that advances microbial electrosynthesis systems toward commercialization