Annika C. Mosier

Relative abundance and diversity of ammonia‐oxidizing archaea and bacteria in the San Francisco Bay estuary

Ammonia oxidation in marine and estuarine sediments plays a pivotal role in the cycling and removal of nitrogen. Recent reports have shown that the newly discovered ammonia-oxidizing archaea can be both abundant and diverse in aquatic and terrestrial ecosystems. In this study, we examined the abundance and diversity of ammonia-oxidizing archaea (AOA) and betaproteobacteria (beta-AOB) across physicochemical gradients in San Francisco Bay--the largest estuary on the west coast of the USA. In contrast to reports that AOA are far more abundant than beta-AOB in both terrestrial and marine systems, our quantitative PCR estimates indicated that beta-AOB amoA (encoding ammonia monooxygenase subunit A) copy numbers were greater than AOA amoA in most of the estuary. Ammonia-oxidizing archaea were only more pervasive than beta-AOB in the low-salinity region of the estuary. Both AOA and beta-AOB communities exhibited distinct spatial structure within the estuary. AOA amoA sequences from the north part of the estuary formed a large and distinct low-salinity phylogenetic group. The majority of the beta-AOB sequences were closely related to other marine/estuarine Nitrosomonas-like and Nitrosospira-like sequences. Both ammonia-oxidizer community composition and abundance were strongly correlated with salinity. Ammonia-oxidizing enrichment cultures contained AOA and beta-AOB amoA sequences with high similarity to environmental sequences. Overall, this study significantly enhances our understanding of estuarine ammonia-oxidizing microbial communities and highlights the environmental conditions and niches under which different AOA and beta-AOB phylotypes may thrive.

Core and Intact Polar Glycerol Dibiphytanyl Glycerol Tetraether Lipids of Ammonia-Oxidizing Archaea Enriched from Marine and Estuarine Sediments

ABSTRACT Glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary (“Candidatus Nitrosoarchaeum limnia”), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and “Candidatus Nitrosopumilus maritimus”; however, they were absent in the group I.1b AOA “Candidatus Nitrososphaera gargensis.”

Spatial Variability in Nitrification Rates and Ammonia-Oxidizing Microbial Communities in the Agriculturally Impacted Elkhorn Slough Estuary, California

ABSTRACT Ammonia oxidation—the microbial oxidation of ammonia to nitrite and the first step in nitrification—plays a central role in nitrogen cycling in coastal and estuarine systems. Nevertheless, questions remain regarding the connection between this biogeochemical process and the diversity and abundance of the mediating microbial community. In this study, we measured nutrient fluxes and rates of sediment nitrification in conjunction with the diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing betaproteobacteria (β-AOB). Sediments were examined from four sites in Elkhorn Slough, a small agriculturally impacted coastal California estuary that opens into Monterey Bay. Using an intact sediment core flowthrough incubation system, we observed significant correlations among NO3 −, NO2 −, NH4 +, and PO4 3+ fluxes, indicating a tight coupling of sediment biogeochemical processes. 15N-based measurements of nitrification rates revealed higher rates at the less impacted, lower-nutrient sites than at the more heavily impacted, nutrient-rich sites. Quantitative PCR analyses revealed that β-AOB amoA (encoding ammonia monooxygenase subunit A) gene copies outnumbered AOA amoA gene copies by factors ranging from 2- to 236-fold across the four sites. Sites with high nitrification rates primarily contained marine/estuarine Nitrosospira-like bacterial amoA sequences and phylogenetically diverse archaeal amoA sequences. Sites with low nitrification rates were dominated by estuarine Nitrosomonas-like amoA sequences and archaeal amoA sequences similar to those previously described in soils. This is the first report measuring AOA and β-AOB amoA abundance in conjunction with 15N-based nitrification rates in estuary sediments.

Denitrifier abundance and activity across the San Francisco Bay estuary.

Over 50% of external dissolved inorganic nitrogen inputs to estuaries are removed by denitrification - the microbial conversion of nitrate to nitrogen gas under anaerobic conditions. In this study, denitrifier abundance, potential rates and community structure were examined in sediments from the San Francisco Bay estuary. Abundance of nirK genes (encoding Cu-containing nitrite reductase) ranged from 9.7 × 10(3) to 4.4 × 10(6) copies per gram of sediment, while the abundance of nirS genes (encoding cytochrome cd1 nitrite reductase) ranged from 5.4 × 10(5) to 5.4 × 10(7) copies per gram of sediment. nirK gene abundance was highest in the riverine North Bay, whereas nirS gene abundance was highest in the more marine Central and South Bays. Denitrification potential (DNP) rate measurements were highest in the San Pablo and Central Bays and lowest in the North Bay. nirS-type denitrifiers may be more biogeochemically important than nirK-type denitrifiers in this estuary, because DNP rates were positively correlated with nirS abundance, nirS abundance was higher than nirK abundance at every site and time point, and nirS richness was higher than nirK richness at every site. Statistical analyses demonstrated that salinity, organic carbon, nitrogen and several metals were key factors influencing denitrification rates, nir abundance and community structure. Overall, this study provides valuable new insights into the abundance, diversity and biogeochemical activity of estuarine denitrifying communities and suggests that nirS-type denitrifiers likely play an important role in nitrogen removal in San Francisco Bay, particularly at high-salinity sites.

Global Biodiversity of Aquatic Ammonia-Oxidizing Archaea is Partitioned by Habitat

Archaea play an important role in nitrification and are, thus, inextricably linked to the global carbon and nitrogen cycles. Since the initial discovery of an ammonia monooxygenase α-subunit (amoA) gene associated with an archaeal metagenomic fragment, archaeal amoA sequences have been detected in a wide variety of nitrifying environments. Recent sequencing efforts have revealed extensive diversity of archaeal amoA sequences within different habitats. In this study, we have examined over 8000 amoA sequences from the literature and public databases in an effort to understand the ecological factors influencing the distribution and diversity of ammonia-oxidizing archaea (AOA), with a particular focus on sequences from aquatic habitats. This broad survey provides strong statistical support for the hypothesis that different environments contain distinct clusters of AOA amoA sequences, as surprisingly few sequences are found in more than one habitat type. Within aquatic environments, salinity, depth in the water column, and temperature were significantly correlated with the distribution of sequence types. These findings support the existence of multiple distinct aquatic AOA populations in the environment and suggest some possible selective pressures driving the partitioning of AOA amoA diversity.

Metabolites Associated with Adaptation of Microorganisms to an Acidophilic, Metal-Rich Environment Identified by Stable-Isotope-Enabled Metabolomics

ABSTRACT Microorganisms grow under a remarkable range of extreme conditions. Environmental transcriptomic and proteomic studies have highlighted metabolic pathways active in extremophilic communities. However, metabolites directly linked to their physiology are less well defined because metabolomics methods lag behind other omics technologies due to a wide range of experimental complexities often associated with the environmental matrix. We identified key metabolites associated with acidophilic and metal-tolerant microorganisms using stable isotope labeling coupled with untargeted, high-resolution mass spectrometry. We observed >3,500 metabolic features in biofilms growing in pH ~0.9 acid mine drainage solutions containing millimolar concentrations of iron, sulfate, zinc, copper, and arsenic. Stable isotope labeling improved chemical formula prediction by >50% for larger metabolites (>250 atomic mass units), many of which were unrepresented in metabolic databases and may represent novel compounds. Taurine and hydroxyectoine were identified and likely provide protection from osmotic stress in the biofilms. Community genomic, transcriptomic, and proteomic data implicate fungi in taurine metabolism. Leptospirillum group II bacteria decrease production of ectoine and hydroxyectoine as biofilms mature, suggesting that biofilm structure provides some resistance to high metal and proton concentrations. The combination of taurine, ectoine, and hydroxyectoine may also constitute a sulfur, nitrogen, and carbon currency in the communities. IMPORTANCE Microbial communities are central to many critical global processes and yet remain enigmatic largely due to their complex and distributed metabolic interactions. Metabolomics has the possibility of providing mechanistic insights into the function and ecology of microbial communities. However, our limited knowledge of microbial metabolites, the difficulty of identifying metabolites from complex samples, and the inability to link metabolites directly to community members have proven to be major limitations in developing advances in systems interactions. Here, we show that combining stable-isotope-enabled metabolomics with genomics, transcriptomics, and proteomics can illuminate the ecology of microorganisms at the community scale. Microbial communities are central to many critical global processes and yet remain enigmatic largely due to their complex and distributed metabolic interactions. Metabolomics has the possibility of providing mechanistic insights into the function and ecology of microbial communities. However, our limited knowledge of microbial metabolites, the difficulty of identifying metabolites from complex samples, and the inability to link metabolites directly to community members have proven to be major limitations in developing advances in systems interactions. Here, we show that combining stable-isotope-enabled metabolomics with genomics, transcriptomics, and proteomics can illuminate the ecology of microorganisms at the community scale.

Seasonal Synechococcus and Thaumarchaeal population dynamics examined with high resolution with remote in situ instrumentation

Monterey Bay, CA is an Eastern boundary upwelling system that is nitrogen limited much of the year. In order to resolve population dynamics of microorganisms important for nutrient cycling in this region, we deployed the Environmental Sample Processor with quantitative PCR assays targeting both ribosomal RNA genes and functional genes for subclades of cyanobacteria (Synechococcus) and ammonia-oxidizing Archaea (Thaumarchaeota) populations. Results showed a strong correlation between Thaumarchaea abundances and nitrate during the spring upwelling but not the fall sampling period. In relatively stratified fall waters, the Thaumarchaeota community reached higher numbers than in the spring, and an unexpected positive correlation with chlorophyll concentration was observed. Further, we detected drops in Synechococcus abundance that occurred on short (that is, daily) time scales. Upwelling intensity and blooms of eukaryotic phytoplankton strongly influenced Synechococcus distributions in the spring and fall, revealing what appear to be the environmental limitations of Synechococcus populations in this region. Each of these findings has implications for Monterey Bay biogeochemistry. High-resolution sampling provides a better-resolved framework within which to observe changes in the plankton community. We conclude that controls on these ecosystems change on smaller scales than are routinely assessed, and that more predictable trends will be uncovered if they are evaluated within seasonal (monthly), rather than on annual or interannual scales.

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.

Spatiotemporal relationships between the abundance, distribution, and potential activities of ammonia-oxidizing and denitrifying microorganisms in intertidal sediments.

The primary objective of this study was to gain an understanding of how key microbial communities involved in nitrogen cycling in estuarine sediments vary over a 12-month period. Furthermore, we sought to determine whether changes in the size of these communities are related to, or indicative of, seasonal patterns in fixed nitrogen dynamics in Elkhorn Slough—a small, agriculturally impacted estuary with a direct connection to Monterey Bay. We assessed sediment and pore water characteristics, abundance of functional genes for nitrification (bacterial and archaeal amoA, encoding ammonia monooxygenase subunit A) and denitrification (nirS and nirK, encoding nitrite reductase), and measurements of potential nitrification and denitrification activities at six sites. No seasonality in the abundance of denitrifier or ammonia oxidizer genes was observed. A strong association between potential nitrification activity and the size of ammonia-oxidizing bacterial communities was observed across the estuary. In contrast, ammonia-oxidizing archaeal abundances remained relatively constant in space and time. Unlike many other estuaries, salinity does not appear to regulate the distribution of ammonia-oxidizing communities in Elkhorn Slough. Instead, their distributions appear to be governed over two different time scales. Long-term niche characteristics selected for the gross size of archaeal and bacterial ammonia-oxidizing communities, yet covariation in their abundances between monthly samples suggests that they respond in a similar manner to short-term changes in their environment. Abundances of denitrifier and ammonia oxidizer genes also covaried, but site-specific differences in this relationship suggest differing levels of interaction (or coupling) between nitrification and denitrification.

(15)N- and (2)H proteomic stable isotope probing links nitrogen flow to archaeal heterotrophic activity.

Understanding how individual species contribute to nutrient transformations in a microbial community is critical to prediction of overall ecosystem function. We conducted microcosm experiments in which floating acid mine drainage (AMD) microbial biofilms were submerged - recapitulating the final stage in a natural biofilm life cycle. Biofilms were amended with either (15)NH4(+) or deuterium oxide ((2)H2O) and proteomic stable isotope probing (SIP) was used to track the extent to which different members of the community used these molecules in protein synthesis across anaerobic iron-reducing, aerobic iron-reducing and aerobic iron-oxidizing environments. Sulfobacillus spp. synthesized (15)N-enriched protein almost exclusively under iron-reducing conditions whereas the Leptospirillum spp. synthesized (15)N-enriched protein in all conditions. There were relatively few (15)N-enriched archaeal proteins, and all showed low atom% enrichment, consistent with Archaea synthesizing protein using the predominantly (14)N biomass derived from recycled biomolecules. In parallel experiments using (2)H2O, extensive archaeal protein synthesis was detected in all conditions. In contrast, the bacterial species showed little protein synthesis using (2)H2O. The nearly exclusive ability of Archaea to synthesize proteins using (2)H2O may be due to archaeal heterotrophy, whereby Archaea offset deleterious effects of (2)H by accessing (1)H generated by respiration of organic compounds.