Enrichment and characterization of ammonia-oxidizing archaea from the open ocean : phylogeny, physiology and stable isotope fractionation

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 5 (2011): 1796–1808, doi:10.1038/ismej.2011.58.Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the north Pacific Ocean that have been maintained in laboratory culture for over three years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S rRNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98-99% identity) to those recovered in DNA and cDNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonium (NH4 +) to nitrite (NO2 -). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (15εNH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal 15εNH3 ranged from 13- 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by 15εNH3 is similar.This work was supported by a Woods Hole Oceanographic Institution (WHOI) Postdoctoral Scholar fellowship to AES and the WHOI Ocean Life Institute

Dual isotope analyses indicate efficient processing of atmospheric nitrate by forested watersheds in the northeastern U.S.

Author Posting. © Springer, 2008. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Biogeochemistry 90 (2008): 15-27, doi:10.1007/s10533-008-9227-2.Nitrogen from atmospheric deposition serves as the dominant source of new nitrogen to forested ecosystems in the northeastern U.S.. By combining isotopic data obtained using the denitrifier method, with chemistry and hydrology measurements we determined the relative importance of sources and control mechanisms on nitrate (NO3-) export from five forested watersheds in the Connecticut River watershed. Microbially produced NO3- was the dominant source (82-100%) of NO3- to the sampled streams as indicated by the δ15N and δ18O of NO3-. Seasonal variations in the δ18O-NO3- in streamwater are controlled by shifting hydrology and temperature affects on biotic processing, resulting in a relative increase in unprocessed NO3- export during winter months. Mass balance estimates find that the unprocessed atmospherically derived NO3- stream flux represents less than 3% of the atmospherically delivered wet NO3- flux to the region. This suggests that despite chronically elevated nitrogen deposition these forests are not nitrogen saturated and are retaining, removing, and reprocessing the vast majority of NO3- delivered to them throughout the year. These results confirm previous work within Northeastern U.S. forests and extend observations to watersheds not dominated by a snow-melt driven hydrology. In contrast to previous work, unprocessed atmospherically derived NO3- export is associated with the period of high recharge and low biotic activity as opposed to spring snowmelt and other large runoff events.This work was funded by an EPA STAR Fellowship (FP-91637501-1) and a grant from QLF/The Sound Conservancy to RTB

Seasonal variations in the nitrogen isotopic composition of settling particles at station K2 in the western subarctic North Pacific

Intensive observations using hydrographical cruises and moored sediment trap deployments during 2010 and 2012 at station K2 in the North Pacific western subarctic gyre (WSG) revealed seasonal changes in δ15N of both suspended and settling particles. Suspended particles (SUS) were collected from depths between the surface and 200 m; settling particles by drifting traps (DST; 100-200 m) and moored traps (MST; 200 and 500 m). All particles showed higher δ15N values in winter and lower in summer, contrary to the expected by isotopic fractionation during phytoplankton nitrate consumption. We suggest that these observed isotopic patterns are due to ammonium consumption via light-controlled nitrification, which could induce variations in δ15N(SUS) of 0.4-3.1 ‰ in the euphotic zone (EZ). The δ15N(SUS) signature was reflected by δ15 N(DST) despite modifications during biogenic transformation from suspended particles in the EZ. δ15 N enrichment (average: 3.6 ‰) and the increase in C:N ratio (by 1.6) in settling particles suggests year-round contributions of metabolites from herbivorous zooplankton as well as TEPs produced by diatoms. Accordingly, seasonal δ15 N(DST) variations of 2.4-7.0 ‰ showed a significant correlation with primary productivity (PP) at K2. By applying the observed δ15 N(DST) vs. PP regression to δ15 N(MST) of 1.9-8.0 ‰, we constructed the first annual time-series of PP changes in the WSG. Moreover, the monthly export ratio at 500 m was calculated using both estimated PP and measured organic carbon fluxes. Results suggest a 1.6 to 1.8 times more efficient transport of photosynthetically-fixed carbon to the intermediate layers occurs in summer/autumn rather than winter/spring

Denitrification likely catalyzed by endobionts in an allogromiid foraminifer

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 6 (2012): 951–960, doi:10.1038/ismej.2011.171.Nitrogen can be a limiting macronutrient for carbon uptake by the marine biosphere. The process of denitrification (conversion of nitrate to gaseous compounds, including N2) removes bioavailable nitrogen, particularly in marine sediments, making it a key factor in the marine nitrogen budget. Benthic foraminifera reportedly perform complete denitrification, a process previously considered nearly exclusively performed by bacteria and archaea. If the ability to denitrify is widespread among these diverse and abundant protists, a paradigm shift is required for biogeochemistry and marine microbial ecology. However, to date, the mechanisms of foraminiferal denitrification are unclear and it is possible that the ability to perform complete denitrification is due to symbiont metabolism in some foraminiferal species. Using sequence analysis and GeneFISH, we show that for a symbiont-bearing foraminifer, the potential for denitrification resides in the endobionts. Results also identify the endobionts as denitrifying pseudomonads and show that the allogromiid accumulates nitrate intracellularly, presumably for use in denitrification. Endobionts have been observed within many foraminiferal species, and in the case of associations with denitrifying bacteria, may provide fitness for survival in anoxic conditions. These associations may have been a driving force for early foraminiferal diversification, which is thought to have occurred in the Neoproterozoic when anoxia was widespread.This research was supported by NSF grant EF-0702491 to JMB, KLC and VPE; some ship support was provided by NSF MCB-0604084 to VPE and JMB.2012-06-0

Integrated metatranscriptomic and metagenomic analyses of stratified microbial assemblages in the open ocean

As part of an ongoing survey of microbial community gene expression in the ocean, we sequenced and compared ~38 Mbp of community transcriptomes and ~157 Mbp of community genomes from four bacterioplankton samples, along a defined depth profile at Station ALOHA in North Pacific subtropical gyre (NPSG). Taxonomic analysis suggested that the samples were dominated by three taxa: Prochlorales, Consistiales and Cenarchaeales, which comprised 36–69% and 29–63% of the annotated sequences in the four DNA and four cDNA libraries, respectively. The relative abundance of these taxonomic groups was sometimes very different in the DNA and cDNA libraries, suggesting differential relative transcriptional activities per cell. For example, the 125 m sample genomic library was dominated by Pelagibacter (~36% of sequence reads), which contributed fewer sequences to the community transcriptome (~11%). Functional characterization of highly expressed genes suggested taxon-specific contributions to specific biogeochemical processes. Examples included Roseobacter relatives involved in aerobic anoxygenic phototrophy at 75 m, and an unexpected contribution of low abundance Crenarchaea to ammonia oxidation at 125 m. Read recruitment using reference microbial genomes indicated depth-specific partitioning of coexisting microbial populations, highlighted by a transcriptionally active high-light-like Prochlorococcus population in the bottom of the photic zone. Additionally, nutrient-uptake genes dominated Pelagibacter transcripts, with apparent enrichment for certain transporter types (for example, the C4-dicarboxylate transport system) over others (for example, phosphate transporters). In total, the data support the utility of coupled DNA and cDNA analyses for describing taxonomic and functional attributes of microbial communities in their natural habitats.Gordon and Betty Moore FoundationUnited States. Dept. of EnergyNational Science Foundation (U.S.) (Science and Technology Center Award EF0424599

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments

Community shift of ammonia-oxidizing bacteria along an anthropogenic pollution gradient from the Pearl River Delta to the South China Sea

The phylogenetic diversity and abundance of ammonia-oxidizing beta-proteobacteria (beta-AOB) was analyzed along an anthropogenic pollution gradient from the coastal Pearl River Delta to the South China Sea using the ammonia monooxygenase subunit A (amoA) gene. Along the gradient from coastal to the open ocean, the phylogenetic diversity of the dominant genus changed from Nitrosomonas to Nitrosospira, indicating the niche specificity by these two genera as both salinity and anthropogenic influence were major factors involved. The diversity of bacterial amoA gene was also variable along the gradient, with the highest in the deep-sea sediments, followed by the marshes sediments and the lowest in the coastal areas. Within the Nitrosomonas-related clade, four distinct lineages were identified including a putative new one (A5-16) from the different sites over the large geographical area. In the Nitrosospira-related clade, the habitat-specific lineages to the deep-sea and coastal sediments were identified. This study also provides strong support that Nitrosomonas genus, especially Nitrosomonas oligotropha lineage (6a) could be a potential bio-indicator species for pollution or freshwater/wastewater input into coastal environments. A suite of statistical analyses used showed that water depth and temperature were major factors shaping the community structure of beta-AOB in this study area