Alyson E. Santoro

Isotopic Signature of N2O Produced by Marine Ammonia-Oxidizing Archaea

Archaea may account for the majority of marine nitrous oxide emissions to the atmosphere. The ocean is an important global source of nitrous oxide (N2O), a greenhouse gas that contributes to stratospheric ozone destruction. Bacterial nitrification and denitrification are thought to be the primary sources of marine N2O, but the isotopic signatures of N2O produced by these processes are not consistent with the marine contribution to the global N2O budget. Based on enrichment cultures, we report that archaeal ammonia oxidation also produces N2O. Natural-abundance stable isotope measurements indicate that the produced N2O had bulk δ15N and δ18O values higher than observed for ammonia-oxidizing bacteria but similar to the δ15N and δ18O values attributed to the oceanic N2O source to the atmosphere. Our results suggest that ammonia-oxidizing archaea may be largely responsible for the oceanic N2O source.

Denitrifier Community Composition along a Nitrate and Salinity Gradient in a Coastal Aquifer

ABSTRACT Nitrogen flux into the coastal environment via submarine groundwater discharge may be modulated by microbial processes such as denitrification, but the spatial scales at which microbial communities act and vary are not well understood. In this study, we examined the denitrifying community within the beach aquifer at Huntington Beach, California, where high-nitrate groundwater is a persistent feature. Nitrite reductase-encoding gene fragments (nirK and nirS), responsible for the key step in the denitrification pathway, were PCR amplified, cloned, and sequenced from DNAs extracted from aquifer sediments collected along a cross-shore transect, where groundwater ranged in salinity from 8 to 34 practical salinity units and in nitrate concentration from 0.5 to 330 μM. We found taxonomically rich and novel communities, with all nirK clones exhibiting <85% identity and nirS clones exhibiting <92% identity at the amino acid level to those of cultivated denitrifiers and other environmental clones in the database. Unique communities were found at each site, despite being located within 40 m of each other, suggesting that the spatial scale at which denitrifier diversity and community composition vary is small. Statistical analyses of nir sequences using the Monte Carlo-based program ∫-Libshuff confirmed that some populations were indeed distinct, although further sequencing would be required to fully characterize the highly diverse denitrifying communities at this site.

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

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 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98–99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH3) to nitrite (NO2−). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect (15ɛNH3) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal 15ɛNH3 ranged from 13‰ to 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 15ɛNH3 is similar.

Interactions between fire and bark beetles in an old growth pine forest

Abstract Management strategies for old growth pine forests have recently begun to include prescribed burns. Fire could influence interactions between bark beetles and mature pine trees, but we cannot predict the effects because we know too little about the numeric and functional responses of bark beetle populations to fire, and because we do not know how fire affects the oleoresin defense system of pine trees. We estimated population abundance of Ips spp. (Coleoptera: Scolytidae), and the resin flow of mature red pines (Pinus resinosa), before and after a prescribed burn, inside and outside the burn, in an old growth forest at Itasca State Park, Minnesota. Following a prescribed burn in April, the local abundance of Ips pini increased by two-fold during May, decreased by a comparable amount during 6 weeks starting in mid-July, and was otherwise unchanged. The abundance of I. grandicollis and I. perroti were unaffected, while that of a specialist predator, Thanasimus dubius (Coleoptera: Cleridae) increased by 30–90% during May. Many mature trees that sustained no visible crown damage from the fire were attacked by Ips within the scorched region of the lower bole. Oleoresin flow increased substantially in trees with scorched boles, which may limit the probability that trees will be killed by bark beetles following a ground fire. We tested whether fire increases the probability that a healthy tree will sustain bark beetle attacks by locating beetle-infested trees inside and outside the burned area, and comparing their growth history (from growth rings) with paired, unattacked trees. Surprisingly, there was no indication of recently declining growth, or chronically slow growth, in beetle-infested trees, either inside or outside the prescribed burn. Half of the trees attacked by Ips in 1998 were dead in 1999 and the remainder were partly girdled by the attacks, which increases their subsequent vulnerability to fires, insects, and pathogens. Ips bark beetles can exert meaningful effects on the survivorship of red pine populations, and their demographic impact is probably increased by ground fires.

Genomic and proteomic characterization of “Candidatus Nitrosopelagicus brevis”: An ammonia-oxidizing archaeon from the open ocean

Significance Thaumarchaeota are among the most abundant microbial cells in the ocean, but to date, complete genome sequences for marine Thaumarchaeota are lacking. Here, we report the 1.23-Mbp genome of the pelagic ammonia-oxidizing thaumarchaeon “Candidatus Nitrosopelagicus brevis” str. CN25. We present the first proteomic data, to our knowledge, from this phylum, which show a high proportion of proteins translated in oligotrophic conditions. Metagenomic fragment recruitment using data from the open ocean indicate the ubiquitous presence of Ca. N. brevis-like sequences in the surface ocean and suggest Ca. N. brevis as a model system for understanding the ecology and evolution of pelagic marine Thaumarchaeota. Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name “Candidatus Nitrosopelagicus brevis” str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

Ecophysiology of uncultivated marine euryarchaea is linked to particulate organic matter

Particles in aquatic environments host distinct communities of microbes, yet the evolution of particle-specialized taxa and the extent to which specialized microbial metabolism is associated with particles is largely unexplored. Here, we investigate the hypothesis that a widely distributed and uncultivated microbial group—the marine group II euryarchaea (MGII)—interacts with living and detrital particulate organic matter (POM) in the euphotic zone of the central California Current System. Using fluorescent in situ hybridization, we verified the association of euryarchaea with POM. We further quantified the abundance and distribution of MGII 16 S ribosomal RNA genes in size-fractionated seawater samples and compared MGII functional capacity in metagenomes from the same fractions. The abundance of MGII in free-living and >3 μm fractions decreased with increasing distance from the coast, whereas MGII abundance in the 0.8–3 μm fraction remained constant. At several offshore sites, MGII abundance was highest in particle fractions, indicating that particle-attached MGII can outnumber free-living MGII under oligotrophic conditions. Compared with free-living MGII, the genome content of MGII in particle-associated fractions exhibits an increased capacity for surface adhesion, transcriptional regulation and catabolism of high molecular weight substrates. Moreover, MGII populations in POM fractions are phylogenetically distinct from and more diverse than free-living MGII. Eukaryotic phytoplankton additions stimulated MGII growth in bottle incubations, providing the first MGII net growth rate measurements. These ranged from 0.47 to 0.54 d−1. However, MGII were not recovered in whole-genome amplifications of flow-sorted picoeukaryotic phytoplankton and heterotrophic nanoflagellates, suggesting that MGII in particle fractions are not physically attached to living POM. Collectively, our results support a linkage between MGII ecophysiology and POM, implying that marine archaea have a role in elemental cycling through interactions with particles.

Nitrogen cycling in the secondary nitrite maximum of the eastern tropical North Pacific off Costa Rica

Nitrite is a central intermediate in the marine nitrogen cycle and represents a critical juncture where nitrogen can be reduced to the less bioavailable N2 gas or oxidized to nitrate and retained in a more bioavailable form. We present an analysis of rates of microbial nitrogen transformations in the oxygen deficient zone (ODZ) within the eastern tropical north Pacific ocean (ETNP). We determined rates using a novel one-dimensional model using the distribution of nitrite and nitrate concentrations, along with their natural abundance nitrogen (N) and oxygen (O) isotope profiles. We predict rate profiles for nitrate reduction, nitrite reduction, and nitrite oxidation throughout the ODZ, as well as the contributions of anammox to nitrite reduction and nitrite oxidation. Nitrate reduction occurs at a maximum rate of 25 nM d−1 at the top of the ODZ, at the same depth as the maximum rate of nitrite reduction, 15 nM d−1. Nitrite oxidation occurs at maximum rates of 10 nM d−1 above the secondary nitrite maximum (SNM), but also in the secondary nitrite maximum, within the ODZ. Anammox contributes to nitrite oxidation within the ODZ, but cannot account for all of it. Nitrite oxidation within the ODZ that is not through anammox is also supported by microbial gene abundance profiles. Our results suggest the presence of nitrite oxidation within the ETNP ODZ, with implications for the distribution and physiology of marine nitrite-oxidizing bacteria, and for total nitrogen loss in the largest marine ODZ.

Diverse, uncultivated bacteria and archaea underlying the cycling of dissolved protein in the ocean

Dissolved organic nitrogen (DON) supports a significant amount of heterotrophic production in the ocean. Yet, to date, the identity and diversity of microbial groups that transform DON are not well understood. To better understand the organisms responsible for transforming high molecular weight (HMW)-DON in the upper ocean, isotopically labeled protein extract from Micromonas pusilla, a eukaryotic member of the resident phytoplankton community, was added as substrate to euphotic zone water from the central California Current system. Carbon and nitrogen remineralization rates from the added proteins ranged from 0.002 to 0.35 μmol C l−1 per day and 0.03 to 0.27 nmol N l−1 per day. DNA stable-isotope probing (DNA-SIP) coupled with high-throughput sequencing of 16S rRNA genes linked the activity of 77 uncultivated free-living and particle-associated bacterial and archaeal taxa to the utilization of Micromonas protein extract. The high-throughput DNA-SIP method was sensitive in detecting isotopic assimilation by individual operational taxonomic units (OTUs), as substrate assimilation was observed after only 24 h. Many uncultivated free-living microbial taxa are newly implicated in the cycling of dissolved proteins affiliated with the Verrucomicrobia, Planctomycetes, Actinobacteria and Marine Group II (MGII) Euryarchaeota. In addition, a particle-associated community actively cycling DON was discovered, dominated by uncultivated organisms affiliated with MGII, Flavobacteria, Planctomycetes, Verrucomicrobia and Bdellovibrionaceae. The number of taxa assimilating protein correlated with genomic representation of TonB-dependent receptor (TBDR)-encoding genes, suggesting a possible role of TBDR in utilization of dissolved proteins by marine microbes. Our results significantly expand the known microbial diversity mediating the cycling of dissolved proteins in the ocean.

Assessment of Nitrogen and Oxygen Isotopic Fractionation During Nitrification and Its Expression in the Marine Environment

Nitrification is a microbially-catalyzed process whereby ammonia (NH(3)) is oxidized to nitrite (NO(2)(-)) and subsequently to nitrate (NO(3)(-)). It is also responsible for production of nitrous oxide (N(2)O), a climatically important greenhouse gas. Because the microbes responsible for nitrification are primarily autotrophic, nitrification provides a unique link between the carbon and nitrogen cycles. Nitrogen and oxygen stable isotope ratios have provided insights into where nitrification contributes to the availability of NO(2)(-) and NO(3)(-), and where it constitutes a significant source of N(2)O. This chapter describes methods for determining kinetic isotope effects involved with ammonia oxidation and nitrite oxidation, the two independent steps in the nitrification process, and their expression in the marine environment. It also outlines some remaining questions and issues related to isotopic fractionation during nitrification.

Influence of ammonia oxidation rate on thaumarchaeal lipid composition and the TEX86 temperature proxy

Significance The membrane lipids of marine Archaea form the basis of the temperature proxy called TEX86, which is used for paleoclimate reconstructions from the Jurassic to the present. To date there remains no satisfactory explanation for how planktonic Archaea are able to record water column temperatures, because TEX86 does not correlate well with in situ growth temperatures in the modern ocean. Here we show that the TEX86 lipid ratio changes in response to cellular growth rate, which is controlled by the ammonia oxidation rate. This implies that variation in the TEX86 ratio with water depth is influenced by the metabolic activity of Thaumarchaeota in the water column. Archaeal membrane lipids known as glycerol dibiphytanyl glycerol tetraethers (GDGTs) are the basis of the TEX86 paleotemperature proxy. Because GDGTs preserved in marine sediments are thought to originate mainly from planktonic, ammonia-oxidizing Thaumarchaeota, the basis of the correlation between TEX86 and sea surface temperature (SST) remains unresolved: How does TEX86 predict surface temperatures, when maximum thaumarchaeal activity occurs below the surface mixed layer and TEX86 does not covary with in situ growth temperatures? Here we used isothermal studies of the model thaumarchaeon Nitrosopumilus maritimus SCM1 to investigate how GDGT composition changes in response to ammonia oxidation rate. We used continuous culture methods to avoid potential confounding variables that can be associated with experiments in batch cultures. The results show that the ring index scales inversely (R2 = 0.82) with ammonia oxidation rate (ϕ), indicating that GDGT cyclization depends on available reducing power. Correspondingly, the TEX86 ratio decreases by an equivalent of 5.4 °C of calculated temperature over a 5.5 fmol·cell−1·d−1 increase in ϕ. This finding reconciles other recent experiments that have identified growth stage and oxygen availability as variables affecting TEX86. Depth profiles from the marine water column show minimum TEX86 values at the depth of maximum nitrification rates, consistent with our chemostat results. Our findings suggest that the TEX86 signal exported from the water column is influenced by the dynamics of ammonia oxidation. Thus, the global TEX86–SST calibration potentially represents a composite of regional correlations based on nutrient dynamics and global correlations based on archaeal community composition and temperature.