Metaproteomics reveals differential modes of metabolic coupling among ubiquitous oxygen minimum zone microbes Alyse K. Hawleya, Heather M. Brewerb, Angela D. Norbeckb, Ljiljana Paša-Tolicb, and Steven J. Hallama,c,d,1 aDepartment of Microbiology and Immunology, cGraduate Program in Bioinformatics, and dGenome Sciences and Technology Training Program, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; and bBiological and Computational Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352 Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved June 10, 2014 (received for review November 26, 2013) Marine oxygen minimum zones (OMZs) are intrinsic water column transformations in nonsulfidic OMZs, providing evidence for features arising from respiratory oxygen demand during organic a cryptic sulfur cycle with the potential to drive inorganic carbon matter degradation in stratified waters. Currently OMZs are expand- fixation processes (13). Indeed, many of the key microbial players ing due to global climate change with resulting feedback on marine implicated in nitrogen and sulfur transformations in OMZs, in- ecosystem function. Here we use metaproteomics to chart spatial cluding Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, and temporal patterns of gene expression along defined redox and SUP05/ARCTIC96BD-19 Gammaproteobacteria have the gradients in a seasonally stratified fjord to better understand metabolic potential for inorganic carbon fixation (14–19), and microbial community responses to OMZ expansion. The expres- previous process rate measurements in OMZs point to high sion of metabolic pathway components for nitrification, anaerobic rates of dark primary production (20–23).However,therelative ammonium oxidation (anammox), denitrification, and inorganic contribution of each player to coupled carbon (C), nitrogen carbon fixation were differentially expressed across the redoxcline (N), and sulfur (S) biogeochemistry as a function of redox and covaried with distribution patterns of ubiquitous OMZ microbes zonation and in response to perturbation remains to be de- including Thaumarchaeota, Nitrospina, Nitrospira, Planctomycetes, termined. These contributions have important implications and SUP05/ARCTIC96BD-19 Gammaproteobacteria. Nitrification and for understanding long-term ecological and biogeochemical inorganic carbon fixation pathways affiliated with Thaumarchaeota impacts of OMZ expansion and intensification on marine carbon dominated dysoxic waters, and denitrification, sulfur oxidation, and and nutrient cycling. inorganic carbon fixation pathways affiliated with the SUP05 group Here we investigate changes in microbial community structure of nitrate-reducing sulfur oxidizers dominated suboxic and anoxic and function in a seasonally stratified fjord, Saanich Inlet on waters. Nitrifier nitrite oxidation and anammox pathways affili- Vancouver Island British Columbia Canada, to better understand ated with Nirospina, Nitrospira, and Planctomycetes, respectively, metabolic coupling along defined redox gradients. We combine also exhibited redox partitioning between dysoxic and suboxic cultivation-independent molecular approaches including small waters. The numerical abundance of SUP05 proteins mediating subunit ribosomal RNA gene pyrosequencing, metagenomics, inorganic carbon fixation under anoxic conditions suggests that and metaproteomics to chart the progression of microbial com- SUP05 will become increasingly important in global ocean carbon munity structure and gene expression along the redoxcline. We then and nutrient cycling as OMZs expand. construct a conceptual model linking different modes of inorganic Significance arine oxygen (O2) minimum zones (OMZs) are wide- Mspread and naturally occurring water column features that arise when respiratory O2 demand during decomposition of or- Oxygen is an important organizing principle in marine ecosys- ganic matter exceeds O2 availability in stratified waters. Opera- tems. As oxygen levels decline, energy is increasingly diverted tionally defined by dissolved O2 concentrations <20 μM, OMZs away from higher trophic levels into microbial community promote the use of alternative terminal electron acceptors (TEAs) metabolism causing changes in carbon and nutrient cycling. in microbial energy metabolism that results in climate active gas Here we use metagenomic and metaproteomic methods to production including carbon dioxide (CO2), nitrous oxide (N2O), chart in situ metabolic networks linking key microbial players and methane (CH4) (1). Currently, OMZs constitute ∼7% of the driving carbon and nutrient cycling in a seasonally stratified ocean volume (1, 2). However, global warming promotes con- fjord, Saanich Inlet, a model ecosystem for studying microbial ditions for OMZ expansion and intensification, e.g., reduced O2 responses to changing levels of water column oxygen de- solubility and increased stratification, with resulting feedback ficiency. Based on this evidence, we develop a conceptual ontheclimatesystem(3,4). model that describes coupling of chemotrophic energy production − − Within OMZs, the use of nitrate (NO3 ) and nitrite (NO2 )as with dark carbon fixation along defined redox gradients with TEAs in dissimilatory nitrate reduction (denitrification) and implications for primary production and possibly carbon sedi- anaerobic ammonium oxidation (anammox) results in fixed ni- mentation in expanding marine oxygen minimum zones. trogen loss in the form of N2O and dinitrogen gas (N2), re- spectively (5, 6). Because OMZs account for up to 50% of Author contributions: A.K.H., L.P.-T., and S.J.H. designed research; A.K.H. and H.M.B. oceanic N2 production, they have the potential to limit primary performed research; A.K.H. and A.D.N. analyzed data; A.K.H. and S.J.H. wrote the paper; production in overlying surface waters (7, 8). A recent model and S.J.H. supervised the project. suggests that nitrogen fixation in proximity to OMZ waters can The authors declare no conflict of interest. balance nitrogen loss processes (9), and several studies along This article is a PNAS Direct Submission. redoxclines in the Eastern Tropical South Pacific and Baltic Sea Data deposition: The sequence reported in this paper has been deposited in the NCBI Bio- have measured nitrogen fixation rates that support a close spatial Project database, www.ncbi.nlm.nih.gov/bioproject (BioProject no. 247822; accession nos. coupling between nitrogen loss and nitrogen fixation consistent SAMN02781345–SAMN02781359). 1 with this model (10–12). Moreover, recent studies have begun To whom correspondence should be addressed. Email: [email protected]. SCIENCES to link the oxidation of reduced sulfur-compounds including This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ENVIRONMENTAL 2- thiosulfate (S2O3 ) and hydrogen sulfide (H2S) to nitrogen 1073/pnas.1322132111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322132111 PNAS | August 5, 2014 | vol. 111 | no. 31 | 11395–11400 Downloaded by guest on September 29, 2021 carbon fixation with distributed nitrogen and sulfur-based energy creased in abundance within the SNTZ and SZ. Similar trends metabolism. were observed with respect to ORF counts and NSAF values (Tables S2–S4). The nitrite oxidizing bacterium Nitrospina gra- Results and Discussion cilis (26), mediating the second step of nitrification, was abun- Water Column Chemistry and Molecular Sampling. To evaluate changes dant in UO and LO samples and decreased in abundance within in water column redox gradients associated with different stages the SNTZ and SZ. A second nitrite oxidizing bacterium of stratification and renewal, samples were collected from the Nitrospira defluvii (18), although absent from pyrotag datasets, Saanich Inlet water column from station S3 on April 9, 2008 exhibited high NSAF values with a similar distribution pattern as (Apr08) and from multiple stations from the inlet mouth (S4) Thaumarchaeota and N. gracilis. Anammox bacteria affiliated midpoint (S3) and end (S2) on September 1, 2009 (Sep09) (Fig. with the Planctomycetes (Tables S2–S4) exhibited intermediate S1A). Water column chemistry profiles indicated four redox zones: abundance (∼1%) in the UO and LO samples, decreasing in upper oxycline (UO), lower oxycline (LO), sulfide nitrate transition abundance within the SNTZ before increasing again in the SZ. zone (SNTZ), and sulfidic zone (SZ) (Fig. 1 and Fig. S1B), Planctomycete ORF abundance increased along the redoxcline, whereas protein NSAF values were high in the UO and LO, generally corresponding to dysoxic (20–90 μmol O2), suboxic decreasing to intermediate values within the SNTZ and SZ. (1–20 μmol O2), anoxic (<1 μmol O2), and anoxic sulfidic con- ditions (2) (Fig. S1B). Water column redox zonation and asso- These patterns of protein expression confirm previous reports of ciated microbial community structure was consistent with other coupled nitrification and anammox observed in OMZs based on OMZs (2, 24), making Saanich Inlet a tractable model ecosystem process rate and functional marker gene abundance (27, 28). for studying microbial community responses to changing levels In addition to known players in the nitrogen cycle, taxa in- of water column oxygen deficiency. volved in sulfur cycling or coupled nitrogen and sulfur cycling To explore changes in microbial community structure and were also abundant and active in the water column. Multiple function