Metagenome of a Versatile Chemolithoautotroph from Expanding Oceanic Dead Zones

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Science  23 Oct 2009:
Vol. 326, Issue 5952, pp. 578-582
DOI: 10.1126/science.1175309

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Oxygen minimum zones, also known as oceanic “dead zones,” are widespread oceanographic features currently expanding because of global warming. Although inhospitable to metazoan life, they support a cryptic microbiota whose metabolic activities affect nutrient and trace gas cycling within the global ocean. Here, we report metagenomic analyses of a ubiquitous and abundant but uncultivated oxygen minimum zone microbe (SUP05) related to chemoautotrophic gill symbionts of deep-sea clams and mussels. The SUP05 metagenome harbors a versatile repertoire of genes mediating autotrophic carbon assimilation, sulfur oxidation, and nitrate respiration responsive to a wide range of water-column redox states. Our analysis provides a genomic foundation for understanding the ecological and biogeochemical role of pelagic SUP05 in oxygen-deficient oceanic waters and its potential sensitivity to environmental changes.

Dissolved oxygen (O2) concentration is a critical determinant of marine ecosystem structure and function. O2 deficiency results in habitat compression and reduced productivity for aerobic organisms, with concomitant expansion of conditions favoring chemolithotrophic energy metabolism (1), which results in nitrogen loss and production of climate-active trace gases such as nitrous oxide (N2O) and methane (CH4) (2, 3). Extensive oxygen minimum zones (OMZs), defined by O2 concentrations < 20 μM, are found throughout the eastern North Pacific (ENP), eastern South Pacific (ESP), northern Indian Ocean, and southwest African shelf waters (3). Moreover, climate change–induced expansion and intensification of OMZs is occurring globally, with potentially deleterious effects on oceanic nitrogen cycling and carbon sequestration (1, 46).

Although the extent of oxygen deficiency varies among OMZs, taxonomic surveys reveal conserved patterns of microbial community composition (79). In all sites examined, small subunit ribosomal RNA (SSU rRNA) gene libraries are enriched with sequences affiliated with chemoautotrophic gill symbionts of deep-sea clams and mussels (ESP-OMZ-sequence-accumulation–1 (EOSA-1) in the ESP and gamma-proteobacterial sulfur oxidizer (GSO) cluster in African shelf waters) (911). Phylogenetic analyses indicate that the GSO–EOSA-1 complex consists of two closely related, cooccurring and uncultivated lineages, ARCTIC96BD-19 (12) and SUP05 (13), with the latter encompassing the symbionts (Fig. 1, A and B). Blooming SUP05 populations have recently been implicated in chemolithotrophic sulfide (H2S) oxidation coupled to nitrate (NO3) reduction in African shelf waters (10). Both ARCTIC96BD-19 and SUP05 populations are also found in nonsulfidic waters of the ENP and ESP, which suggests that they can adopt alternative modes of energy metabolism. Given the potential importance of ARCTIC96BD-19 and SUP05 to carbon, nitrogen, and sulfur cycling in OMZs, a deeper understanding of the metabolic capabilities of these lineages is needed to constrain their respective ecological and biogeochemical roles.

Fig. 1

(A) Phylogenetic tree of ARCTIC96BD-19 and SUP05 lineages based on comparative SSU rRNA gene analysis. The tree was inferred using maximum likelihood implemented in PHYML. (B) Relative abundance of ARCTIC96BD-19 and SUP05 SSU rRNA sequences recovered from Saanich Inlet (SI), eastern North Pacific (ENP), eastern South Pacific (ESP) (9), and southwest African shelf waters (Namibia) (10).

Saanich Inlet, British Columbia, is a seasonally anoxic fjord characterized by an annual stratification and deep-water renewal cycle (14), resulting in large water-column redox gradients and high rates of trace gas production and consumption (fig. S1, A to C). Previously, we identified pelagic SUP05 in the Saanich Inlet water column, representing up to 37% of total bacteria (table S1) (11). Further examination of SUP05 SSU rRNA gene copy number during seasonal stratification revealed blooming populations below the oxycline, reaching up to 4.75 × 105 copies per ml in regions of H2S and NO3 depletion (15) (fig. S2). High-resolution SSU rRNA gene surveys revealed two SUP05 phylotypes, SI-1 and SI-2 (Figs. 1A and 2A), differing by ~4% nucleotide identity. Although SI-1 dominated suboxic waters year round, SI-2 was less common, and transiently increased during deep-water renewal events, which indicated ecologically differentiated populations (Fig. 2A). Here we analyzed 16 bi-directionally end-sequenced fosmid libraries constructed from environmental DNA samples spanning oxic to anoxic waters over the seasonal stratification and deep-water renewal cycle in order to reconstruct the metabolic potential of uncultivated pelagic SUP05 populations (fig. S1D).

Fig. 2

(A) Phylotype abundance of SUP05 SI-1 (black circles) and SI-2 (white circles) based on recovery of SSU rRNA gene sequences in PCR-generated clone libraries. (B) Mean depth of coverage of the SUP05 metagenome plotted over the Saanich Inlet nitrate profile during the 2006–07 season. Sample dates and depths are noted on the x and y axes, respectively.

To identify SUP05-specific scaffolds, fosmid paired-end sequences were assembled and binned on the basis of shared sequence similarity to symbiont reference genomes (16, 17) and analysis of intrinsic oligonucleotide composition patterns (fig. S3). Nineteen scaffolds encompassing 1.16 million base pairs of SUP05 DNA (SUP05 metagenome) were identified (table S2) and taxonomically verified (table S3). Consistent with SSU rRNA gene survey data, a majority of fosmid paired-end sequences within SUP05 scaffolds were derived from samples exhibiting elevated SI-1 phylotype abundance (Fig. 2B). Of 861 genes shared between symbiont reference genomes, 80% were conserved in the SUP05 metagenome (supporting online material text, Fig. 3 and fig. S4). Many of these genes are predicted to mediate informational processing steps, particularly translation, although a significant fraction function in carbon, sulfur, amino acid, and coenzyme metabolism (figs. S5 and S6). Approximately 35% of gene content predicted in the SUP05 metagenome was unique, including genes implicated in DNA uptake and repair, denitrification, and adaptive or stress responses (15).

Fig. 3

Gene content comparison between SUP05 metagenome and symbiont reference genomes. Nested circles from outermost to innermost represent the following: (i and ii) Functional predictions based on clusters of orthologous groups (COG) on the forward and reverse strands of the R. magnifica reference genome (see fig. S4 for COG color code designation). (iii) Conservation of gene content and (iv) genes conserved in symbionts, but absent from the SUP05 metagenome. (Inset) Venn diagram depicts predicted gene distribution among SUP05 metagenome and symbiont reference genomes. Values correspond to the number of shared genes among overlapping genomes, with each genome used as the original query. The dotted line represents the open-genome configuration of the SUP05 metagenome. *The discrepancy in core size when SUP05 metagenome is used as query (774) compared with symbionts (~683) reflects gene content redundancy in the metagenome assembly.

Similar to symbiotic counterparts, the SUP05 metagenome harbors genes mediating the Calvin-Benson-Bassham (CBB) cycle for autotrophic carbon assimilation, including a single form II ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) gene, which both implicate SUP05 in chemosynthetic carbon fixation (18). In addition, a gene encoding β-class carbonic anhydrase, a potential CO2-concentrating mechanism, was also identified. The repertoire of genes required for converting fixed carbon to hexose and ribose sugars via gluconeogenesis and the nonoxidative branch of the pentose phosphate pathway were identified, along with the majority of tricarboxylic acid (TCA) cycle components (fig. S6). However, we did not recover genes for enzymes mediating the interconversion of succinyl-CoA and 2-oxoglutarate (fig. S6), which indicated that SUP05 is potentially an obligate autotroph as posited earlier for clam symbionts (16, 17).

From the standpoint of energy metabolism, the SUP05 metagenome harbors a diverse repertoire of genes involved in chemolithotrophic oxidation of reduced sulfur compounds. We found genes encoding flavocytochrome c sulfide dehydrogenase (fccAB) and sulfide quinone oxidoreductase (sqr), mediating oxidation of H2S to elemental sulfur (S0) (fig. S6). The presence of two enzymatic complexes may facilitate sulfur-based energy metabolism under variable sulfide regimes. In addition, dissimilatory sulfite reductase subunits (dsrAB), adenosine 5′-phosphosulfate (APS) reductase (apr), and adenosine 5′-triphosphate (ATP) sulfurylase (sat) mediating complete oxidation of S0 to sulfate, and the Sox pathway (soxABXYZ) for thiosulfate (S2O32−) oxidation (fig. S6) (19) were conserved between pelagic SUP05 and symbiont reference genomes. The capacity to obtain electrons from S2O32− may be of ecological relevance, given that mixing of sulfidic and oxygenated water masses results in S2O32− accumulation owing to chemical oxidation of H2S (20, 21). Moreover, the apparent absence of soxCD sulfur dehydrogenase genes indicates the capacity to store S0, which can be subsequently oxidized via the reverse dissimilatory sulfate reduction (DSR) pathway and thereby provisions SUP05 in the absence of ambient reductant (22). Indeed, soxCD homologs are also absent from symbiont reference genomes, and colloidal sulfur globule formation has been associated with a subset of clam symbionts (23).

Although symbiont reference genomes harbor multiple aerobic respiratory complexes (16, 17), none were recovered in the SUP05 metagenome, consistent with a facultative or strictly anaerobic life-style. Indeed, all enzymatic machinery needed to reduce NO3 to nitrous oxide (N2O), including membrane-bound (narKK2GHJI) and periplasmic (napFBAHGD) dissimilatory nitrate reductases, which potentially operate under high and low NO3 conditions, respectively (24, 25); copper-containing nitrite reductase (nirK); and N2O forming nitric oxide reductase (norCB) (Fig. 4 and fig. S6), were identified, which mechanistically implicated pelagic SUP05 in biological nitrogen loss from oxygen-deficient oceanic waters. Moreover, the genomic colocalization of sulfur oxidation and denitrification genes in close proximity to Crp/Fnr transcriptional regulators (Fig. 4) may allow coordinated gene expression in response to changing redox states or nutrient limitation (24, 26, 27).

Fig. 4

Alignment of an ungapped region of a SUP05 metagenomic scaffold, encoding genes involved in nitrate and sulfur metabolism, with the corresponding genomic regions of symbiont reference genomes. The height of red bars corresponds to nucleotide similarity over conserved genomic regions. Proper scaffold assembly across this region was verified by full-length sequencing of two overlapping fosmids.

We identified more than 10 putative toxin-antitoxin (TA) modules in the SUP05 metagenome, which indicated a highly regulated stress response (table S4). TA modules consist of a stable toxin and a labile antitoxin and are commonly associated with environmental bacteria, where they control induction of reversible cellular stasis (28). Of specific interest is a TA module of the RelE superfamily identified within an operon encoding molybdopterin-guanine dinucleotide synthase (mobA) (Fig. 4). The product of MobA, molybdopterin-guanine dinucleotide (MGD), is an essential cofactor for all described classes of nitrate reductase (29), and therefore, mobA expression is integral to denitrification. In this regard, the integration of a TA system into a denitrification regulon may allow SUP05 to persist during periods of extreme NO3 limitation, analogous to other forms of nutritional stress response (e.g., amino acid starvation in Escherichia coli) (30).

As the number of studies surveying OMZ community structure increases, the ubiquity and abundance of SUP05 becomes apparent. Analysis of the SUP05 metagenome, together with water-column disposition of pelagic SUP05 with respect to H2S and NO3 gradients, resolves a chemolithoautotrophic metabolism based on oxidation of reduced sulfur compounds with NO3 through multiple and highly regulated bioenergetic routes. Although pelagic distribution of SUP05 in Saanich Inlet and African shelf waters is predicated on the basis of vertical H2S and NO3 gradients, the presence of this lineage in nonsulfidic OMZs (e.g., ENP and ESP) emphasizes the need to more fully resolve the chemical speciation of sulfur compounds with respect to SUP05 physiology and population structure. Moreover, the contribution of SUP05-mediated denitrification to the present unbalanced nitrogen cycle and the potential interplay with anaerobic ammonia-oxidizing bacteria within sulfidic and nonsulfidic OMZs remains to be investigated.

Paradoxically, as “dark” primary producers, blooming SUP05 populations have the potential to fix CO2 while simultaneously producing N2O. As habitat range increases with OMZ expansion and intensification, this role will only become more visible and significant. Therefore, the SUP05 metagenome provides a functional template for analysis of gene expression in relation to climatologically relevant biogeochemical transformations within oxygen-deficient oceanic waters. This information should prove useful in the development of monitoring tools to assess microbial community responses to OMZ expansion and intensification.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S7

Tables S1 to S4


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. This work was performed under the auspices of the U.S. Department of Energy's Office of Science, Biological, and Environmental Research Program and by the University of California, Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory under contract no. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under contract no. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract no. DE-AC02-06NA25396. This work was also supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada 328256-07 and STPSC 356988, Canada Foundation for Innovation (CFI) 17444; Canadian Institute for Advanced Research (CIFAR), and the Center for Bioinorganic Chemistry (CEBIC). D.A.W. was supported by NSERC, Killam Trust, and the Tula Foundation–funded Centre for Microbial Diversity and Evolution (CMDE). We thank M. Robert (Institute of Ocean Sciences, Sidney, BC, Canada), C. Payne, L. Pakhomova, and J. Granger (UBC) for help in sampling and chemical analyses and the captains and crews of the CCGS John P. Tulley and HMS John Strickland for logistical support. We thank the Joint Genome Institute, including K. Barry, S. Pitluck, and E. Kirton, for technical assistance and A. Page, K. Mitchell, and S. Lee in the Hallam laboratory for reading the manuscript. This metagenome project has been deposited at the DNA DataBank of Japan and European Molecular Biology Laboratory, and GenBank, under the project accession ACSG00000000. The version described in this paper is the first version, ACSG01000000. SSU rRNA gene sequences were deposited at GenBank under the accession numbers GQ345343-GQ351265, and fosmid sequences were deposited under the accession numbers GQ351266 to GQ351269 and GQ369726.
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