Sulfur Oxidation Genes in Diverse Deep-Sea Viruses

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Science  16 May 2014:
Vol. 344, Issue 6185, pp. 757-760
DOI: 10.1126/science.1252229

Virus-Enhanced Sulfur Oxidation

How do microbial viruses affect subsurface microbial communities? Anantharaman et al. (p. 757, published online 1 May) investigated the interactions between ubiquitous marine lithotrophs found at hydrothermal vents and their viruses. The genes for sulfur oxidation in viruses that infect abundant marine chemosynthetic sulfur-oxidizing bacteria enhanced sulfur oxidation, thereby influencing the biogeochemical sulfur cycle.


Viruses are the most abundant biological entities in the oceans and a pervasive cause of mortality of microorganisms that drive biogeochemical cycles. Although the ecological and evolutionary effects of viruses on marine phototrophs are well recognized, little is known about their impact on ubiquitous marine lithotrophs. Here, we report 18 genome sequences of double-stranded DNA viruses that putatively infect widespread sulfur-oxidizing bacteria. Fifteen of these viral genomes contain auxiliary metabolic genes for the α and γ subunits of reverse dissimilatory sulfite reductase (rdsr). This enzyme oxidizes elemental sulfur, which is abundant in the hydrothermal plumes studied here. Our findings implicate viruses as a key agent in the sulfur cycle and as a reservoir of genetic diversity for bacterial enzymes that underpin chemosynthesis in the deep oceans.

Chemolithoautotrophic bacteria are ubiquitous in the dark oceans (1), where they serve as a sink for CO2 (2) through primary production that equals up to 53% of the particulate organic carbon exported from the photic zone (3). Uncultured sulfur-oxidizing bacteria of the SUP05 clade are among the most abundant and widespread marine chemolithoautotrophs, fixing carbon and oxidizing reduced sulfur species and hydrogen in diverse marine environments such as hydrothermal vent plumes (4), hydrothermal vent-associated animals (5, 6), and oxygen minimum zones (7), where they underpin cryptic links between the sulfur and nitrogen cycles (8). Although viruses are abundant in these deep-sea ecosystems (9), little is known about viruses that infect lithotrophic primary producers.

We conducted shotgun metagenomic sequencing on samples from five different hydrothermal vent plumes and associated deep ocean waters at the Eastern Lau Spreading Center (Lau Basin) in the western Pacific Ocean and one plume at Guaymas Basin in the Gulf of California (10, 11) (table S1). De novo assembly of sequence reads and binning by tetranucleotide signatures (12) revealed discrete genomic “bins” (fig. S1). Five bins (henceforth Lau77, Lau85, Lau87, Lau218, and Lau220) contained 18 viral genome sequences of putative SUP05 viruses. Phylogeny of the viral large terminase gene (terL) (fig. S2) [which reflects phage DNA packaging mechanisms (13)], synteny with well-characterized phages of known taxonomy (fig. S3), and results of protein sequence similarity searches against public sequence databases (fig. S4) indicated that the five viruses belonged to three marine viral families of the orders Caudovirales (double-stranded DNA viruses, no RNA stage), Podoviridae, Siphoviridae, and Myoviridae (table S2).

Fifteen of the 18 viral genomes (from four of the five SUP05 viral genomic bins) contained genes encoding the α (rdsrA) and γ (rdsrC) subunits of the reverse-acting dissimilatory sulfite reductase (Rdsr) complex for elemental sulfur oxidation (Fig. 1). No other rdsr genes or other sulfur oxidation genes were present on the viral genomes. Analysis of bacterial genome bins recovered from Lau and Guaymas metagenomes revealed colocalized rdsr genes in the order rdsrABEFHCMKLJOPN in the Gammaproteobacteria Lau10 (SUP05), Lau62 (EC-01-9C-26), and Lau60 (unclassified). The Deltaproteobacterium Lau20 (Sar324) (14) possessed only rdsrAB. Regions flanking the bacterial rdsr genes showed no similarity to the viral genome sequences, suggesting that viral rdsr genes were derived from selective retention rather than recent homologous recombination with bacterial genomic DNA.

Fig. 1 Gene content of 15 phage genomes from three viral families retrieved from Lau and Guaymas basins.

Colored nested circles represent syntenous viral genomes and contiguous genomic fragments from locations indicated in the legend. Gray boxes on the outermost circles indicate predicted genes from the Kilo Moana viruses. rdsrA and rdsrC genes are highlighted in yellow. Kbp, thousand base pairs.

Phylogenetic analyses indicated that all viral rdsrA genes recovered were affiliated with SUP05 Gammaproteobacteria (74 to 96% amino acid identity) (fig. S5) and distinct from rdsrA genes of other bacteria (Fig. 2). We identified two distinct groups of rdsrA sequences that each included both viral and bacterial sequences. All viral rdsrA genes fall into group one, except for Lau85, which contained two copies of rdsrA with one representative in each group. Bacterial representatives of group one included the SUP05 GB-1 and GB-2 from Guaymas, as well as Bathymodiolus mussel symbionts (6). Group two was populated by SUP05 from oxygen minimum zones (7) and symbionts of deep-sea clams (5). The tight phylogenetic clustering of rdsrA gene sequences of three distinct phage families with SUP05 bacteria in two separate lineages suggests that the phage rdsrA genes originated from SUP05 and were transferred to viruses. These observations are analogous to those of core photosynthesis genes in cyanobacterial phages and other microbe-derived auxiliary metabolic genes (15, 16) (e.g., psbA, psbD, mazG) that are similar but not identical to known hosts, forming subclusters distinct from host proteins (17, 18).

Fig. 2 Phylogeny of rdsrA genes from Lau and Guaymas basins inferred by Maximum Likelihood.

(A) Phylogenetic tree showing the SUP05 rdsrA gene clade in relation to closely related sequences. (B) Detailed view of the SUP05 rdsrA clade. Group one and group two subclades are shown on the right. Sequences are colored by geographical origin: blue, Guaymas Basin; red, Kilo Moana (Lau Basin); green, Tahi Moana (Lau Basin); purple, ABE (Lau Basin); brown, Tui Malila (Lau Basin); orange, Mariner (Lau Basin).

The amino acid sequences deduced from the viral rdsrA and rdsrC genes indicated the capacity to serve as functional sulfur-oxidizing enzymes. Phage RdsrA contained all conserved sulfite reductase residues and secondary structure elements for α helix and β sheets (fig. S6). Similarly, a multiple alignment of RdsrC amino acid sequences indicated highly conserved residues across two distinct groups (fig. S7). We also identified other genes in the viral genomes with high sequence identity to SUP05, including iron-sulfur cluster proteins, cytochromes, and sulfur relay proteins (table S3). The existence of these additional SUP05-like genes on viral genomes supports their specificity to SUP05 bacteria and suggests a role for viral genes in supplementing host metabolism.

Elemental sulfur is a key and stable intermediate of bacterial oxidation of reduced sulfur compounds and constitutes a “bottleneck” in the sulfur cycle (19). Sulfur-oxidizing bacteria with an incomplete Sox pathway, such as SUP05, form intracellular or extracellular globules of elemental sulfur that serve as an electron-donor reserve for energy metabolism (Fig. 3) (4, 1921). The presence of rdsrA and rdsrC on viral genomes may offer selective advantages to the viruses by supplementing host pathways for oxidation of this sulfur during infection. First, enhanced expression of rdsrA could replenish proteins involved in a rate-limiting reaction in the host, as previously demonstrated with cyanobacterial phage D1 proteins involved in photosynthesis (22). Second, phage rdsrC could maintain or increase high transcription levels to ensure efficient delivery of sulfur substrate to the RdsrAB complex during infection. Thus, phage auxiliary metabolic genes that can supplement or sustain sulfur oxidation metabolism in their hosts may ensure continued viral infection and replication.

Fig. 3 Schematic of the sulfur oxidation pathway in SUP05 bacteria.

The gray box indicates the reaction affected by SUP05 viruses. Key genes are shown in italics: sqr (sulfide quinone reductase), sox (sulfur oxidation), rdsr (reverse dissimilatory sulfite reductase), apr (adenosine 5′-phosphosulfate reductase), and sat (sulfate adenylyltransferase).

A coupled bioenergetic-thermodynamic reaction path model (23) indicated that aerobic elemental sulfur oxidation potentially accounted for 80 to 93% of the total lithotrophic energy available in the ABE and Mariner hydrothermal plumes at Lau Basin at two temperatures that were representative of the plumes studied here (Fig. 4A and fig. S8). The model predicted a minor role for 19 other lithotrophic microbial metabolisms, including oxidation of other reduced sulfur species such as hydrogen sulfide, sulfite, and thiosulfate. These results were consistent with a previous model of hydrothermal plumes that found elemental sulfur to be the most abundant source of chemosynthetic energy (24), as well as with our previous work (4, 25, 26), which was based on the same approach. Our model predicted greater relative energy yields for more energetically favored metabolisms, such as sulfur oxidation, because we assumed that metabolic reactions occurred in sequence from most to least energetically favored. Our model also enabled reactions to explicitly modify the product and reactant pools. Although there is some uncertainty regarding the speciation of sulfur present in the plume, elemental sulfur is a central intermediate in the oxidation of other reduced forms of sulfur used by SUP05 (Fig. 3). X-ray fluorescence maps showed that sulfur was abundant in the plumes (Fig. 4B and fig. S8), whereas microprobe x-ray diffraction showed that elemental sulfur was widely present in particle aggregates with other crystalline phases, such as pyrite (Fig. 4C and fig. S8). Although we did not conclusively identify intracellular elemental sulfur, our results showed that elemental sulfur presented an abundant source of energy for SUP05 bacteria in hydrothermal plumes and deep ocean waters of Guaymas and Lau basins.

Fig. 4 Bioenergetics and occurrence of sulfur in the ABE hydrothermal plume.

(A) Modeled free energies of catabolic reactions as a percentage of total available free energy in the ABE hydrothermal plume at 2.5° and 5°C. Total available free energy in the plume is normalized per kilogram of plume fluid (p.f.) and per kilogram of vent fluid (v.f.). (B) Distribution of iron (red) and sulfur (green) in particles collected at 0.5 m above the ABE vent. Locations where elemental sulfur was detected by microprobe x-ray diffraction measurements are indicated as spots 0, 1, 2, and 19. (C) Radially integrated diffractograms with elemental sulfur peaks annotated (asterisks) at 22.0°, 25.7°, and 34.1° 2θ.

The abundance and diversity of viruses infecting SUP05 bacteria in hydrothermal plumes suggests that chemolithoautotrophs in the deep sea face viral predation pressures similar to photosynthetic microbes in the surface waters (27). The marked synteny and conservation of the four viruses studied here (95 to 99% genome nucleotide identity) across hydrothermal vent environments and ocean basins suggests that these viruses are ubiquitous in marine environments dominated by SUP05 bacteria. Analyses of the Pacific Ocean Virome data set (28) (fig. S9), which notably contains viral communities from oxygen minimum zones dominated by SUP05 (7), revealed the presence of rdsrA and rdsrC genes (table S5), consistent with the prevalence of phage-encoded sulfur oxidation beyond hydrothermal plumes and in the wider pelagic oceans.

To date, deep-sea SUP05 has evaded growth in laboratory cultures; thus, direct host-phage manipulations and validation of the underlying mechanisms of phage-influenced sulfur oxidation remain a challenge. Yet, this study demonstrates the sequence-based elucidation of microbial community dynamics through the discovery of phages that infect a widespread deep-sea bacterium. The existence of rdsr genes in viral genomes reveals a mechanism for horizontal transfer of genes associated with sulfur cycling (29) and implicates viruses in the evolutionary dynamics of a central step in the planetary cycling of sulfur.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S10

Tables S1 to S6

References (3078)

Data S1

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: This project is funded in part by the Gordon and Betty Moore Foundation (grant GBMF2609) and the NSF (grants OCE1038006, OCE1038055, OCE1037991, and OCE1029242). We thank the University of Michigan Rackham Graduate School Faculty Research Fellowship Program for their support and M. Li and S. Jain for their assistance. DNA sequencing was conducted at the University of Michigan DNA Sequencing Core. We acknowledge the captain and crew of R/V Thomas G. Thompson, Chief Scientist A.-L. Reysenbach, and J. Sylvan for sampling and logistical support. For assistance with synchrotron measurements at BL10.3.2, we thank K. J. Edwards, J. V. Sorensen, M. A. Marcus, and S. C. Fakra. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy (DOE) under contract no. DE-AC02-05CH11231. The nucleotide sequences are available from DOE JGI-IMG/MER (Taxon Object IDs: Kilo Moana, 3300001680; ABE, 3300001681; Mariner, 3300001678; Tahi Moana, 3300001679; Tui Malila, 3300001676; Guaymas, 3300001683) and the National Center for Biotechnology Information (BioProject: PRJNA234377). We declare no competing financial interests.
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