Metaproteomics Reveals Differential Modes of Metabolic Coupling Among Ubiquitous Oxygen Minimum Zone Microbes

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), respectively (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 conditions (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 along water column redox gradients, we analyzed paired lineages affiliated with SAR11 within the Alphaproteobacteria metagenomic and metaproteomic datasets from Apr08 and mediating dimethylsulfoniopropionate (DMSP) oxidation (29) paired small subunit ribosomal RNA gene pyrosequencing and were abundant in the UO and LO samples, decreasing in abun- metaproteomics datasets from Sep09 (Fig. S1B). Sanger end dance within the SNTZ and SZ (Fig. S2). Similar trends were sequencing of small insert clone libraries from the three Apr08 observed with respect to ORF counts and NSAF values (Tables S2–S4). Multiple lineages affiliated with SUP05/ARCTIC96BD-19 samples yielded a total of 54,701 ORFs, with an average of – 18,234 ORFs per sample. Small subunit ribosomal RNA (SSU and symbiont-related Gammaproteobacteria (Tables S2 S4) mediating oxidation of reduced sulfur compounds using O rRNA) gene pyrosequencing of the 12 Sep09 samples yielded − 2 87,138 sequences that clustered into 3,385 nonsingleton opera- (19) or NO3 (16), as TEAs were also abundant. The ORFs for ARCTIC96BD-19, SUP05, and symbionts exhibited reciprocal tional taxonomic units at the 97% identity threshold. Tandem distribution patterns, with ARCTIC96BD-19 ORFs decreasing and MS-coupled LC (LC/MS/MS) metaproteomic sequencing iden- SUP05 and symbiont ORFs increasing in abundance within the tified a total of 5,019 unique proteins, a number comparable to SNTZ and SZ. A similar pattern was observed with respect to previous marine metaproteomic studies (25). A consistent NSAF values, with high SUP05 NSAF values in the LO, SNTZ, number of proteins were identified across the Sep09 samples, and SZ. These distribution patterns support previous reports averaging 695 unique proteins per sample (Table S1). Although of ARCTIC96BD-19 and SUP05 population structure (2, 16, variability in protein detection in the Apr08 samples was con- 19, 30, 31). siderable, the high number of unique proteins detected in the Collectively, Thaumarchaeota, Nitrospina, Nitrospira, Planc- Apr08 200-m sample (4,344) enabled identification of more tomycetes, SAR11, SUP05/ARCTIC96BD-19, and symbiont- complete metabolic pathways. related Gammaproteobacteria comprised on average 48% of pyrotag, 41% of metagenomic, and 64% of metaproteomic Patterns of Redox-Driven Niche Partitioning. To determine patterns datasets (Tables S2 and S3). Several taxonomic groups that were of redox-driven niche partitioning, we compared community abundant based on pyrotags (>1%) including Marine Group II composition with ORF counts and protein normalized spectral Euryarchaea, Crenarchaeota, Acidomicrobiales, Bacteroidetes, abundance factors (NSAF; SI Methods) between UO, LO, Chloroflexi, Flavobacteria, and Desulfobacteraceae, and candi- SNTZ, and SZ. Hierarchical clustering of NSAF values was date divisions OD1, OP11, Marine Group A, and SBR1093 were consistent with redox zonation (Fig. 1). Clear trends in protein not well represented in metagenomic or metaproteomic datasets abundance were observed in relation to redox zonation not (Fig. S2). Lack of indigenous reference genomes likely caused reflected in pyrotag and metagenomic datasets, consistent with many sequences originating from these groups to be classified as alternative forms of coupling or regulated gene expression (Fig. no hit or below cutoff (Methods). Consistent with this observa- S2). Ammonia oxidizing Thaumarchaeota, mediating the first tion, BLAST queries against the Genomic Encyclopedia of step of nitrification, dominated UO and LO samples and de- Bacteria and Archaea Microbial Dark Matter (GEBA-MDM) single-cell genome collection (32) yielded only 23 additional protein sequences, which had otherwise been classified as below cutoff or no hit. Conversely, several taxonomic groups including 100 S4_100 S3_100 upper N. defluvii and ARCTIC96BD-19 that were absent in pyrotag 86 oxycline 100 S4_130 datasets exhibited intermediate ORF counts and NSAF values. 100 S3_130 lower oxycline This discrepancy was likely due to incomplete taxonomic reso- S2_100 S / N

bp lution within the Greengenes database. Approximately 1% of 56 S3_150 transition 87 S2_130 Sulfidic pyrotag and 10% of metagenomic and metaproteomic datasets S4_150 zone 42 remained unaffiliated with any taxonomic group. Taken together, 100 S2_150 O2 these results indicate that active nitrogen and sulfur cycling micro- 82 S4_190 - NO3 organisms are the primary contributors to both genetic potential 87 S3_200 H2S S2_200 and gene expression along the redoxcline in Saanich Inlet. 35030025020015010050 0 0 20 50 80 µ Height M Differential Gene Expression Patterns. To investigate patterns of Fig. 1. Hierarchical clustering of metaproteome by NSAF (see Methods)for gene expression driving carbon and energy metabolism along the detected proteins from Sep09 S2, S3, and S4 indicating compartments of the redoxcline in Saanich Inlet, we identified nitrification, anammox, − water column, with adjacent sparklines for oxygen (O2), nitrate (NO3 ), and denitrification, sulfur oxidation, and inorganic carbon fixation hydrogen sulfide (H2S) for each sample. pathway components in metagenomic and metaproteomic

11396 | www.pnas.org/cgi/doi/10.1073/pnas.1322132111 Hawley et al. Downloaded by guest on September 29, 2021 datasets using BLAST. By summing the NSAF values for each N. gracilis, and N. defluvii NXR could confound BLAST-based component, we observed differential patterns of gene expression taxonomic assignment. and metabolic coupling along the redoxcline (Fig. 2). Expression Proteins mediating the partial denitrification pathway from of these pathways was remarkably stable under similar redox SUP05 including dissimilatory nitrate reductase subunits G and conditions in space (Sep09 S2-S4) and time (Apr08 to Sep09; H (Nar), periplasmic nitrate reductase subunits A and B (Nap), Figs. S3–S5). and nitrite reductase (NirK) were detected in the UO increasing Expressed pathways for nitrogen-based energy metabolism in abundance along the redoxcline (Fig. 2 and Fig. S3). Protein progressed from ammonia oxidation and nitrification in the UO NSAF values for SUP05 Nar increased relative to Planctomy- and LO to denitrification in the SNTZ and SZ (Fig. 2 and Fig. cetes NXR in the SNTZ and SZ. Additional proteins for SUP05 S3). Proteins catalyzing the first step of nitrification, ammonia nitric oxide reductase subunits B and C (Nor) were detected with similar NSAF values in the LO, SNTZ, and SZ. Although de- monooxygenase subunits B and C (Amo), from Thaumarchaeota, nitrification pathway components from other taxonomic groups were detected in the UO and LO, decreasing along the redoxcline. were detected in the water column, SUP05 was the only group Proteins catalyzing the second step of nitrification, nitrite oxidase to express consecutive proteins in the denitrification pathway, (NXR), from N. graclilis and N. defluvii followed the same pat- making up 50% of total NSAF values for all denitrification tern of expression as Amo. Moreover, the detection of both Amo proteins (SI Text). These observations point to SUP05 as the and NXR from nitrifying taxa, albeit at lower NSAF values in the − dominant player in nitrogen-based energy metabolism in the SNTZ and SZ, supports recent observations of NO2 oxidation − SNTZ and SZ. The detection of SUP05 Nap and NirK in the UO in the Namibian OMZ with implications for NO3 supply for and LO where O2 concentrations approached 120 μM was un- denitrification (28, 33). Nitrite oxidase from Planctomycetes expected given that 20 μMO2 is a commonly accepted threshold (Fig. 2) (14) had the highest NSAF values of any protein in the for denitrification (34) and may have implications for the O2 UO and LO and exhibited a similar expression profile to Amo threshold for nitrogen loss processes in other OMZs. Addition- and NXR originating from N. gracilis and N. defluvii (Fig. 2 ally, detection of SUP05 Nor and absence of nitrous oxide re- and Fig. S3). Conversely, proteins catalyzing anammox, includ- ductase (NosZ) in the LO and low abundance in SNTZ and SZ ing hydrazine and hydroxylamine oxidoreductases (Anx) from point to SUP05 as a source of N2O. Recent observations of Planctomycetes (Fig. 2 and Fig. S3), exhibited opposing expres- enrichment of genes which encode Nor and NosZ on particles sion patterns, with low NSAF values in the UO and LO that within OMZs suggest a distributed denitrification pathway across increased in the SNTZ and SZ. Contrasting patterns of NXR particle and nonparticle niches (35) and may account for low and Anx expression from Planctomycetes could reflect a meta- NSAF values observed for NorCB and NosZ. Expressed pathways for sulfur-based energy metabolism were bolic response to O , resulting in a shift between maintenance 2 detected in the UO and increased in NSAF value along the energy production in the UO and LO to anammox for growth redoxcline (Fig. 2 and Fig. S4A). Proteins catalyzing sulfide ox- under more favorable redox conditions in the SNTZ and SZ. idation predominantly originated from SUP05/ARCTIC96BD- Alternatively, close sequence similarity between Planctomycetes, 19 and symbiont-related Gammaproteobacteria. With the exception of ARCTIC96BD-19 adenylylsulfate reductase (Apr), the vast majority of proteins originated from SUP05 and sym- Apr08 Sep09 bionts. With respect to SUP05, flavocytochrome C (Fcc), sulfide Nitrogen Sulfur Carbon Nitrogen Sulfur Carbon oxidation proteins (Sox), dissimilatory sulfate reductase (Dsr), and Apr were detected in the UO and increased in NSAF value along the redoxcline. In addition, SUP05 ATP sulfurylase (Sat) Amo Anx Anx Amo Sqr Fcc Sox Dsr Apr Sat 3HP-4HB CBB rACoA Nap NirS NirK Nor NosZ HAO-like Sqr Fcc Sox Dsr Apr Sat 3HP-4HB CBB rACoA Nar NirK NRX Nar NosZ NirS Nap HAO-like Nor 100 m NRX 100 m and sulfide:quinone oxidoreductase (Sqr) were detected in the Thaumarchaeota LO, SNTZ, and SZ, and SNTZ and SZ, respectively. These Nitrospina gracilis Nitrospira defluvii results are consistent with recent SUP05 protein expression Planctomycetes ARCTIC96BD-19 profiles observed in hydrothermal plume and overlying waters SUP05 Symbionts (25). With the exception of Sox, symbiont proteins catalyzing Other 120 m 120 m sulfide oxidation followed the same expression pattern as Thaumarchaeota Nitrospina gracilis SUP05. The expression of sulfur oxidation pathway components Nitrospira defluvii Planctomycetes from SUP05/ARCTIC96BD-19 in the UO and LO is consistent ARCTIC96BD-19 with a cryptic sulfur cycle. However, no proteins from defined SUP05 2− Symbionts sulfate (SO4 )-reducing bacteria were identified in the meta- Other 150 m proteome (13). This observation could reflect a bias against Thaumarchaeota 2− Nitrospina gracilis particle-associated microorganisms capable of SO4 reduction Nitrospira defluvii Planctomycetes during sample processing or the use of alternative electron ARCTIC96BD-19 SUP05 donors including DMSP, elemental sulfur, thiosulfate, or poly- Symbionts Other sulfide in the UO and LO. Additionally, proteins with BLAST hits 200 m 200 m to hydrogenase subunit HupL originating from Guaymas Basin Thaumarchaeota Nitrospina gracilis SUP05 metagenomes (36) were detected in the SNTZ, with NSAF Nitrospira defluvii Planctomycetes values comparable to SUP05 NapA (Fig. S4B), expanding the ARCTIC96BD-19 SUP05 range of potential substrates for SUP05 energy metabolism in the Symbionts Other Saanich Inlet water column. Expressed proteins for three inorganic carbon fixation path- Scale: Metaproteome NSAF upper S/N transition metagenome ways including the 3-hydroxypropionate/4-hydroxybutyrate (3HP- <0.01 0.1 1 5 9 oxycline zone abundance lower sulfidic 4HB) from Thaumarchaeota, reductive acetyl-CoA (rACoA) from oxycline zone Planctomycetes, and Calvin Benson Basham (CBB) cycle from Fig. 2. Distribution and NSAF value of proteins involved in nitrogen and SUP05 were differentially expressed along the redoxcline (Fig. 2 sulfur-based energy metabolism and inorganic carbon fixation for taxa and Fig. S5). Unlike proteins mediating nitrogen and sulfur- abundant in the metaproteome. For metagenome (gray, Apr08 only) and based energy metabolism, ORFs encoding carbon fixation path- metaproteome in upper oxycline (green), lower oxycline (teal), S/N transition way components were found in higher relative abundance in the

zone (blue), and sulfidic zone (purple). See Table S4 for full list of protein metagenome (Fig. 2). SCIENCES

names; Anx indicates anammox hydroxylamine oxidoreductase and hydra- Proteins catalyzing the 3HP-4HB pathway were detected pre- ENVIRONMENTAL zine oxidoreductase proteins. dominantly in the UO including 4-hydroxybutytyl-CoA dehydratase,

Hawley et al. PNAS | August 5, 2014 | vol. 111 | no. 31 | 11397 Downloaded by guest on September 29, 2021 acetyl-CoA carboxylase, and propionyl CoA carboxylase (15, Protein NSAF values for CbbM, a RuBisCO subunit located in 17, 37). Similar expression patterns were observed for Amo and proximity to the nar gene cluster, increased between the UO and other ammonia oxidation pathway components (SI Text), pro- LO and remained relatively constant in the SNTZ and SZ. These viding evidence of inorganic carbon fixation coupled to ammonia results provide functional evidence in support of previous ge- oxidation by Thaumarchaeota in the UO. Consistent with pre- nomic observations positing a highly integrated and redox- vious reports, proteins catalyzing a putative Planctomycete sensitive energy metabolism in SUP05 with direct implications for rACoA pathway were detected in the SZ in Apr08 along with energy supply to inorganic carbon fixation. In addition to co- Anx proteins providing evidence for inorganic carbon fixation ordinated Fcc, Sqr, Nar, Nap, and CbbM expression, an ORF coupled to anammox under sulfidic conditions (2.1 μM) (38). encoding a hydroxylamine-oxidoreductase homolog (HAO-like) Protein NSAF values for SUP05 CBB pathway components in- located in the nap/fcc gene cluster was among the most abundant creased relative to other bacteria in the SNTZ and SZ, providing SUP05 proteins detected in the SZ (Figs. 2 and 3 and Fig. S3). compelling evidence for inorganic carbon fixation coupled to SUP05 hao is closely related to genes found in sulfur-oxidizing sulfide-oxidation and partial denitrification by SUP05. Indeed, endosymbionts, as well as the anammox bacterium Candidatus CBB pathway components had the highest ORF counts and Kuenenia stuttgartiensis. All four HAO homologs contain eight protein NSAF values of all carbon fixation pathways, comprising CxxCH multiheme motifs similar to those found in NrfA, a ni- 47% of all carbon fixation proteins within the SNTZ and SZ. In trite reductase catalyzing dissimilatory nitrate reduction to am- addition to inorganic carbon fixation pathways, the abundance of monia (DNRA) (40). SAR11 DNA and protein in the UO and LO (Fig. S2 and Tables S2 and S3) suggest that heterotrophic remineralization of dis- Metabolic Coupling Model. Although bulk inorganic carbon fixa- solved organic matter (DOM) is an active process in the UO and tion rates within OMZs have been measured (20–23), few studies LO. Specifically, ABC transporter proteins for uptake of glycine have directly linked inorganic carbon fixation with energy me- betaine, spermidine/putrescine, and taurine (sources of carbon, tabolism of defined OMZ microbes (41). With this linkage in nitrogen, and sulfur, respectively) were detected with moderate mind, we construct a metabolic model describing taxonomic and NSAF values within the UO and LO. In addition to consuming metabolic networks coupling pathways of nitrogen- and sulfur- molecular oxygen, remineralization of DOM by SAR11 and based energy metabolism and inorganic carbon fixation along other heterotrophic microbes in the UO and LO could act as the redoxcline in Saanich Inlet based on metaproteomic data- + − a source of NH ,SO2 , and CO . sets (Fig. 4). In this model, heterotrophic remineralization of 4 4 2 − + DOM releases CO ,SO 2 ,andNH within the UO and LO. 2 4 4 + Regulated Gene Expression. Given the numerical abundance of Thaumarchaeota couple oxidation of NH4 to inorganic carbon SUP05, we were able to resolve changes in protein expression fixation via the 3HP-4HB pathway within the UO, producing − originating from a metabolic island integrating nitrogen- and NO2 , a process that has been demonstrated both in culture (42) sulfur-based energy metabolism with inorganic carbon fixation and in situ (43). Nitrous oxide is also produced as a byproduct of + (16) (Fig. 3). Specifically, NSAF values for the SUP05 Sqr, ammonia oxidation (43, 44). Nitrite produced via NH4 oxidation is NarH, and NarG subunits appeared to vary as a function of oxidized in turn by N. defluvii, N. gracilis, and Planctomycetes in O2 concentration, whereas the FccAB and NapAB subunits the UO (14, 28). The extent to which this process is coupled to remained relatively constant in the LO, SNTZ, and SZ (Figs. 2 inorganic carbon fixation within these groups remains to be de- and 3 and Figs. S3 and S4A). Close proximity and similar ex- termined. Ammonia oxidation attenuates as O2 levels decline in pression profiles for napAB and fccAB are consistent with reg- the LO, SNTZ, and SZ, accompanied by a transition to partial ulated gene expression along the redoxcline. Indeed, two ORFs denitrification and anammox. In the LO, SUP05 begins to couple encoding Crp/Fnr transcriptional regulators implicated in redox oxidation of reduced sulfur compounds, and possibly hydrogen, − sensing (39) are located on either side of the nap/fcc gene cluster, with NO3 reduction to N2O to fix inorganic carbon via the CBB with the potential to modulate gene expression, and Crp/Fnr pathway, a trend that increases in the SNTZ and SZ (16, 36, 45, proteins (SUP05_0428) were detected in the Apr08 SZ (Fig. 3). 46). In parallel, Planctomycetes couple anammox to fix inorganic

crp/fnrfccBfccA napFnapB napA napHnapG hao crp/fnr

dsrC-likenarInarJnarH narG narK narK2 cbbO cbbQcbbM

S3_100 * S3_120

Apr 08 S3_200 S4_100 S3_100 S4_130 Fig. 3. Relative abundance of SUP05 genes and S2_100 proteins in two overlapping SUP05 fosmid sequen- S3_130 ces (GQ351266 and GQ351267) (16). Metagenome S4_150

Sep 09 (gray, Apr08 only) and metaproteome for the upper S3_150 oxycline (green), lower oxycline (teal), S/N transition S2_130 zone (blue), and sulfidic zone (purple). Selected S2_150 O2 SUP05 genes involved in denitrification (dark gray - S4_200 NO3 shading), sulfide oxidation (black shading), and pu- S3_200 H2S tative hydroxylamine oxidoreductase (diagonal S2_200 lines) are indicated. Protein abundance shown as 0 20 80 summed NSAF values for all detected ORFs with top SUP05_FGYC13F180031 - 46 μM (GQ351266) SUP05_FGYC13J70002-4, 17 - 30 hit to a given SUP05 protein. Metagenome abun- (GQ351267) dances shown as percentage of ORFs with top hit to scale Key NSAF sulfur carbon upper S /N % metagenome a given SUP05 gene with sparklines for oxygen (O2), oxycline transition abundance − nitrogen HAO-like lower nitrate (NO3 ), and hydrogen sulfide (H2S) for each < 0.02 0.05 0.5 1 3 6 oxycline sulfidic sample.

11398 | www.pnas.org/cgi/doi/10.1073/pnas.1322132111 Hawley et al. Downloaded by guest on September 29, 2021 upper lower S / N transition sulfidic oxycline oxycline zone zone

CO 2 DOM CO2 DOM CO2 DOM CO2 DOM 3HP-4HB 3HP-4HB 3HP-4HB 3HP-4HB + + NH4 NH4+ NH4+ NH4 - - - NO CO NO CO NO NO - 3 2 3 2 3 CO2 3 CO2 NH + NH + + + 4 SO 2- 4 SO 2- NH4 2- NH4 2- 4 4 SO4 SO4 N2O N O - 2 - N2O - N2O NO NO - NO NO 2 CO 2 2 2 CO + 2 + CO2 + CO2 + 2 N O NH4 rACoA N O NH4 rACoA N2O NH4 rACoA N2O NH4 rACoA 2 ? 2 ? ? ?

CO N2 N2 CO N2 CO2 N2 2 CO2 2 CBB CBB CBB CBB X-H2 X-H2 X-H2 X-H2 - - - - NO3 NO3 NO3 NO H2 H2 H2 H2 3

- 2- - 2- - 2- - 2- HS SO4 HS SO4 HS SO4 HS SO4

heterotrophic sulfur SAR11 Nitrospira carbon (pathway indicated) Nitrospina Thaumarchaeota Planctomycetes SUP05 uncultured remineralization oxidation clade defluvii / gracilis bacterium carbon fixation nitrogen hydrogen energy coupling transformation oxidation

Fig. 4. Proposed metabolic model based on metaproteomic observations for heterotrophic remineralization (brown) and energetic coupling (yellow dashed lines) of nitrogen (green), sulfur (red), and hydrogen (orange) based chemolithotrophic energy metabolism with carbon fixation (yellow star) for taxa abundant in the metaproteome. Line weight and arrow size indicate magnitude of metabolic activity. Gray lines, activity not occurring under given conditions; light gray taxa, reduced abundance and metabolic activity.

carbon via the rACoA pathway (14), although the broader water model could be used to convert gene abundance into protein column occurrence of this process remains to be determined. abundance or protein production rates, resulting in more accurate Competition between SUP05 and Planctomycetes for oxidants predictions. Based on the evidence provided here, SUP05 will could help explain variations in spatial and temporal dynamics likely play an important role in such a pathway-centric model. of nitrogen loss processes observed in different OMZs. Al- + Indeed, we identified SUP05 as the dominant contributor to ternatively, potential DNRA by SUP05 could supply NH4 for inorganic carbon fixation within OMZs, providing insight into anammox resulting in a cometabolic linkage. Overall, the inter- ocean carbon and nutrient cycling. actions described in the model are dynamic and reflect patterns In the future, global climate models predict expansion and of redox-driven niche partitioning regulating nitrogen loss pro- intensification of OMZs, with concomitant shoaling and stabili- cesses and carbon flux through ubiquitous OMZ microbes. zation of sulfidic zones (38, 48). Such a scenario would provide Energy for inorganic carbon fixation within the LO, SNTZ, an increased habitat for SUP05, supporting inorganic carbon and SZ is derived in large part from denitrification and anammox fixation via direct oxidation of reduced sulfur compounds and nitrogen loss processes, with the balance between these pro- cryptic sulfur cycling in oxygen-deficient waters, resulting in in- cesses impacting energy flow to either SUP05 or Planctomycetes, creased primary production and potentially increased carbon with concomitant feedback on growth rates. The numerical sedimentation (49). Given an estimate of 4.61 × 1018 Lof dominance of SUP05 DNA and protein in the LO, SNTZ, and O2-deficient marine waters (50) and the range of observed dark SZ relative to Planctomycetes suggests that partial denitrification carbon fixation rates from various OMZs of 0.2–2.5 μM/L/d outcompetes anammox from a bioenergetic perspective. Indeed, (20–23), we estimate 0.4–5 Pg carbon/y fixed in OMZs globally. the difference in free energy yield between the two processes This number represents up to 10% of surface primary production ∼ (denitrification coupled to sulfide oxidation yields 3.5 times the [using 48.5 Pg C/y (51)] and will continue to increase with OMZ Gibbs free energy as anammox under standard conditions) is expansion. With 47% of observed carbon fixation proteins consistent with lower cell abundance and biomass for anammox originating from SUP05, we suggest that SUP05 is responsible bacteria, even though anammox is observed more frequently for 0.2–2.4 Pg carbon/y, representing up to 5% of surface pri- than denitrification in many OMZs (34). As OMZs expand, the mary productivity. Although OMZ expansion is a predicted con- contribution of SUP05 to inorganic carbon fixation may have sequence of global warming, negative feedback loops may significant impact on global ocean carbon cycling if sufficient ultimately lead to increased drawdown of atmospheric CO2 energetic substrates are available. However, the fate of carbon driven in large part by blooming SUP05 populations. fixed in OMZ waters is largely unknown, as the balance between carbon transport and heterotrophic remineralization processes Methods remains to be constrained. Sample Collection. Sample collection was carried out on board the MSV John Strickland in Saanich Inlet April 9, 2008, at station S3 (48°35.30 N, 123°30.22 Future Implications. This study represents the first metaproteome W) (Apr08), and September 1, 2009, at station S2 (48°33.106 N, 123°32.081 of an O2-deficient water column encompassing the range of re- W), station S3, and station S4 (48°38.310 N, 123°30.007 W) (Sep09) (Fig. S1). dox conditions, from dysoxic to anoxic sulfidic, found in OMZs Samples for metagenomics, metaproteomics, and SSU rRNA gene pyrose- globally. Although a recent numerical model by Reed and col- quencing were collected as described in Zaikova et al. (24), with the exception leagues attempted to integrate geochemical processes and func- of the 1.0 L Apr08 metaproteomic samples where RNAlater (Ambion) was used tional gene markers in the Arabian Sea OMZ (47), our con- instead of lysis buffer. Multiple depths at all stations and dates were sampled − − + ceptual model uses protein expression to describe differential for NO3 ,NO2 ,NH4 ,andH2S as previously described in ref. 24 and a Sea Bird metabolic coupling among ubiquitous OMZ microbes. The Reed Electronics O2 sensor on conductivity/temperature/depth instrument was used model implicitly assumes reaction rates scale linearly with gene to monitor O2 concentrations. abundance. Thus, the model does not account for biological in-

formation flow from DNA to RNA to protein, a regulated pro- Environmental Metagenomics. Environmental DNA extraction was carried SCIENCES

cess resulting in assembly of pathways driving real world process out as previously described in Hawley et al. (52) and Zakiova et al. (24) (also ENVIRONMENTAL rates. Incorporation of protein expression information into the www.jove.com/video/1161/). Metagenomic samples were sequenced at the

Hawley et al. PNAS | August 5, 2014 | vol. 111 | no. 31 | 11399 Downloaded by guest on September 29, 2021 Department of Energy Joint Genome Institute (Walnut Creek, CA) by Sanger ACKNOWLEDGMENTS. We thank the crew aboard the MSV John Strickland shotgun sequencing. Sequences were annotated and translated into amino for logistical support; David Walsh, Olena Schevchuk, and Elena Zaikova for acid sequences using the FGENESB pipeline from Softberry (www.softberry. assistance with sample collection; Jinshu Yang for assistance with sample preparation; and Charles Howes for assistance with data analysis. We also com/berry.phtml) as described in Walsh et al. (16). Pyrosequencing of Sep09 thank Sean Crowe and all members of the S.J.H. laboratory for helpful samples was carried out as described in Allers et al. (53). comments along the way. We thank the Joint Genome Institute, including Sussanah Tringe, Stephanie Malfatti, and Tijana Glavina del Rio, for Environmental Metaproteomics. Total environmental protein was extracted technical and project management assistance. This work was performed from Stervix filters, and the peptide sequence was determined by MS/MS (52) under the auspices of the US Department of Energy (DOE) Joint Genome Institute supported by the Office of Science of US DOE Contract DE-AC02- (details described in SI Methods). Proteins were identified using SEQUEST, 05CH11231, the Tula Foundation-funded Centre for Microbial Diversity and taxonomy and function for all metagenomic and metaproteomic sequen- and Evolution, the Natural Sciences and Engineering Research Council of ces were assigned using BLASTP, and NSAF values were determined as de- Canada, the Canada Foundation for Innovation, and the Canadian In- scribed in SI Methods. stitute for Advanced Research through grants awarded to S.J.H. Meta- proteomics support came from the intramural research and development Hierarchical Clustering of Metaproteomic Samples. The NSAF values for all program of the W. R. Wiley Environmental Molecular Sciences Laboratory ≥ (EMSL). EMSL is a national scientific user facility sponsored by the US detected proteins with a PPP 0.95 were used in the calculation of a Sorensen DOE’s Office of Biological and Environmental Research and located at distance matrix using PC-ORD software, and a group average method was used the Pacific Northwest National Laboratory operated by Battelle for the for grouping in construction of clusters. US DOE.

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