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

Rate of inward electron flux normalized to total electrode-attached protein, showing effect of riboflavin.

<p>Maximum current responses after fumarate addition (open bars) were normalized to attached protein values to obtain a specific rate of electron transfer (µA/µg protein; n≥3, +/− standard deviation). Closed bars represent maximum current values after addition of 1 µM riboflavin.</p

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

A model for reversible electron transfer through the Mtr respiratory pathway in <i>S. oneidensis</i> MR-1.

<p>(A) Electrons generated at the electrode surface are transferred to MtrC. MtrC then transfers electrons to MtrA by interacting through MtrB. From MtrA electrons are passed to CymA and through the menaquinone pool to a second CymA interacting with FccA. Approximately 85% of inward electron flux is dependent on flow through the menaquinone pool, while 15% relies on transfer from MtrA to FccA. Multi-heme cytochromes are in red and non-heme proteins in blue. (B) Redox potential windows for components involved in electrode-dependent fumarate reduction in <i>S. oneidensis</i>. Dark red lines represent midpoint potentials for specific hemes within CymA or FccA. The yellow line represents the midpoint potential of the FAD cofactor of FccA (data compiled from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Hartshorne1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-FirerSherwood1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Hartshorne2" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Pealing2" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Butt1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Morris1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016649#pone.0016649-Pessanha1" target="_blank">[46]</a>).</p

Single turnover and catalytic voltammetry of WT and Δ<i>fccA</i> thin films attached to electrodes.

<p>Representative cyclic voltammograms (1 mV/s) of fumarate responses of <i>S. oneidensis</i> MR-1 (black trace, no addition; blue trace, 50 mM fumarate) and Δ<i>fccA</i> (olive trace, no addition; red trace, 50 mM fumarate) after 16 hr of attachment to electrodes poised at +0.24 V <i>versus</i> SHE. The redox peak centered at +0.2 V <i>versus</i> SHE is indicative of a redox active species in close proximity to the electrode surface, i.e. <i>c</i>-type cytochromes exposed on the outer membrane.</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

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Riboflavin causes a shift in the potential required for reductive electron flow into Δ<i>cymA</i>.

<p>Representative cyclic voltammograms (1 mV/s) of A) <i>S. oneidensis</i> MR-1 with no additions (dotted line), 50 mM fumarate (dashed line), and 50 mM fumarate +1 µM riboflavin (solid line) for (A) wild type thin films and (B) Δ<i>cymA</i> thin films. (C) Derivative plots showing midpoint potentials for WT (black traces) and Δ<i>cymA</i> (red traces). The midpoint potential for the <i>menC</i> mutant was similar to Δ<i>cymA</i> (see text for details).</p