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Microbial Metatranscriptomics in a Permanent Marine Oxygen

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Microbial Metatranscriptomics in a Permanent Marine Oxygen Environmental Microbiology (2012) 14(1), 23–40 doi:10.1111/j.1462-2920.2010.02400.x Microbial metatranscriptomics in a permanent marine oxygen minimum zoneemi_2400 23..40 Frank J. Stewart,1 Osvaldo Ulloa3 and dominated the transcriptome in the upper three Edward F. DeLong2* depths, representing one in five protein-coding tran- 1School of Biology, Georgia Institute of Technology, scripts at 85 m. In contrast, transcripts matching the 311 Ferst Drive, Atlanta, GA 30332, USA. anammox bacterium Kuenenia stuttgartiensis domi- 2Department of Civil and Environmental Engineering, nated at the core of the OMZ (200 m; 1 in 12 protein- Massachusetts Institute of Technology, Parsons coding transcripts). The distribution of N. maritimus- Laboratory 48, 15 Vassar Street, Cambridge, MA like transcripts paralleled that of transcripts matching 02139, USA. ammonia monooxygenase genes, which, despite 3Departamento de Oceanografía and Centro de being represented by both bacterial and archaeal Investigación Oceanográfica en el Pacífico Sur-Oriental, sequences in the community DNA, were dominated Universidad de Concepción, Casilla 160-C, Concepción, (> 99%) by archaeal sequences in the RNA, suggest- Chile. ing a substantial role for archaeal nitrification in the upper OMZ. These data, as well as those describing other key OMZ metabolic processes (e.g. sulfur oxi- Summary dation), highlight gene-specific expression patterns Simultaneous characterization of taxonomic compo- in the context of the entire community transcriptome, sition, metabolic gene content and gene expression as well as identify key functional groups for taxon- in marine oxygen minimum zones (OMZs) has poten- specific genomic profiling. tial to broaden perspectives on the microbial and bio- geochemical dynamics in these environments. Here, Introduction we present a metatranscriptomic survey of microbial community metabolism in the Eastern Tropical South Oxygen minimum zones (OMZs) play critical roles in Pacific OMZ off northern Chile. Community RNA was marine community structuring and global biogeochemical sampled in late austral autumn from four depths (50, cycling. Forming at intermediate depths (~100–1000 m) in 85, 110, 200 m) extending across the oxycline and into response to high biological oxygen demand and reduced the upper OMZ. Shotgun pyrosequencing of cDNA ventilation, OMZs occur naturally in zones of nutrient-rich yielded 180 000 to 550 000 transcript sequences per upwelling but are also expanding throughout the world’s depth. Based on functional gene representation, tran- oceans as a result of anthropogenic effects, such as scriptome samples clustered apart from correspond- enhanced nutrient run-off and climate change (Stramma ing metagenome samples from the same depth, et al., 2006; Diaz and Rosenberg, 2008). This expansion highlighting the discrepancies between metabolic critically impacts marine ecosystems, as OMZs, in which potential and actual transcription. BLAST-based dissolved O2 often falls below 10 mM, displace oxygen- characterizations of non-ribosomal RNA sequences respiring macroorganisms (e.g. fish) and create anaero- revealed a dominance of genes involved with both bic, microbially dominated communities whose members oxidative (nitrification) and reductive (anammox, exert important effects on marine nitrogen and carbon denitrification) components of the marine nitrogen cycles (Ulloa and Pantoja, 2009). cycle. Using annotations of protein-coding genes as Oxygen minimum zone communities are typified by a proxies for taxonomic affiliation, we observed depth- low diversity and abundance of pelagic macrofauna but a specific changes in gene expression by key func- complex microbial community adapted to life along the tional taxonomic groups. Notably, transcripts most oxic–anoxic gradient. Notably, OMZ-associated bacteria closely matching the genome of the ammonia- and archaea mediate oceanic fixed nitrogen loss to the oxidizing archaeon Nitrosopumilus maritimus atmosphere through denitrification and the anaerobic oxi- dation of ammonia to N2 (anammox) (Codispoti et al., 2001; Kuypers et al., 2005; Ward et al., 2009). OMZs also Received 26 August, 2010; accepted 11 November, 2010. *For play significant roles in greenhouse gas cycling, for correspondence. E-mail [email protected]; Tel. (+617) 253 5271. example, through the release of the potent heat-trapping © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd 24 F. J. Stewart, O. Ulloa and E. F. DeLong gas nitrous oxide (N2O). Recent genetic and bio- the upper OMZ is intrinsically linked to the dissimilatory geochemical evidence also suggests a role for pelagic nitrogen transformations at the OMZ core, potentially sulfur cycling in OMZs, mediated in part by the dissimila- serving as a vital source of nitrite for anammox and fuel- tory metabolism of sulfur-oxidizing bacteria related to ling an influx of fixed carbon to the system (Lam et al., endosymbionts of deep-sea bivalves (Stevens and Ulloa, 2007; Molina and Farías, 2009). Genetic evidence shows 2008; Lavik et al., 2009; Walsh et al., 2009; Canfield a complex nitrifier community in the ETSP-OMZ com- et al., 2010). posed of both bacteria and archaea, although the relative Studies of the Eastern Tropical South Pacific (ETSP) contributions of these two groups to ammonia-oxidation OMZ off northern Chile and Peru have been critical in remain unclear (Molina et al., 2007; 2010). identifying the organisms and metabolisms characteristic Our knowledge of these diverse OMZ metabolisms is of life in pelagic low oxygen environments. In the ETSP- based largely on studies of individual pathways (e.g. deni- OMZ, persistent upwelling of nutrient-rich waters drives trification, anammox) or taxonomic groups, or on single- high primary production in the photic zone (Daneri et al., gene surveys of phylogenetic (e.g. 16S rRNA) diversity 2000). As photosynthetically derived organic matter sinks, and functional gene abundance (e.g. nitrite reductase, it is respired and degraded by aerobic heterotrophs, ammonia monooxygenase). However, we know little of drawing oxygen down from > 200 mM at the surface to the extent to which specific metabolic processes are less than 1 mM below the oxycline (~50–100 m). Through- represented in the exceedingly diverse pool of genes out the core of the OMZ (~70–500 m), oxygen decreases expressed across a complex microbial community. to nM concentrations, or even to anoxia (Revsbech et al., Community-wide analysis of microbial gene expression in 2009). Oxygen conditions remain depleted throughout the natural communities can help identify unforeseen link- year, creating one of the largest persistently oxygen- ages among metabolic processes (McCarren et al., deficient regions in the global ocean. 2010), as well as inform predictions of the relative syn- As oxygen declines, anaerobic metabolism becomes chrony between metabolic transformations and DNA, increasingly important, significantly altering nutrient and RNA, and protein abundance in diverse microbial assem- organic matter profiles relative to aerobic zones. Notably, blages. Here, we present the first survey of pelagic micro- oxidized nitrogen species dominate as oxidants in dissimi- bial community gene expression (the metatranscriptome) latory respiration by both autotrophs and heterotrophs. in an OMZ, focusing specifically on the ETSP-OMZ off Autotrophic bacteria within the Planctomycetes have northern Chile. been described in the ETSP-OMZ as the primary group High throughout sequencing of the metatranscriptome responsible for anammox, the likely predominant pathway has provided an unprecedented overview of gene expres- for fixed nitrogen loss in this system (Thamdrup et al., sion in natural microbial communities, but thus far has 2006; Hamersley et al., 2007; Galán et al., 2009; Lam been restricted to a handful of aerobic marine environ- et al., 2009). However, heterotrophic denitrification, the ments (Frias-Lopez et al., 2008; Hewson et al., 2009; oxidation of organic matter via a complete sequential 2010; Poretsky et al., 2009; Shi et al., 2009). Here, we reduction of nitrate (NO3)toN2, also occurs in the ETSP- use pyrosequencing to analyse the community RNA and OMZ (Farías et al., 2009), potentially producing ammonia DNA from four depths spanning the aerobic photic zone (via organic matter remineralization) and nitrite (via nitrate (50 m), the oxic–anoxic transition zone (85, 110 m) and reduction) for anammox. Although the range of microor- the anoxic OMZ core (200 m) at a site on the continental ganisms mediating denitrification is not fully described, slope. Using BLAST-based characterizations of protein- the multiple steps of this pathway likely involve diverse coding genes, we characterize dominant patterns in taxonomic groups. Notably, the dissimilatory reduction of metatranscriptome diversity, transcriptional activity and oxidized nitrogen species may involve chemoautotrophic sample relatedness, as well as identify key trends in sulfur-oxidizing bacteria. Indeed, genomic analysis of the taxonomic and functional gene representation. These lineage SUP05, a free-living gammaproteobacterial rela- datasets facilitate comparative analysis of bacterioplank- tive of clam endosymbionts sampled from a North Pacific ton gene expression across diverse oceanic regions, as seasonal OMZ, revealed enzymes necessary for the well as intensive exploration of cryptic, but functionally chemolithotrophic oxidation of reduced sulfur, as well as important, metabolic processes, genes and organisms those for nitrate reduction
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