Community Structure and Function of Planktonic Crenarchaeota: Changes with Depth in the South China Sea

Marine Crenarchaeota represent a widespread and abundant microbial group in marine ecosystems. Here, we investigated the abundance, diversity, and distribution of planktonic Crenarchaeota in the epi-, meso-, and bathypelagic zones at three stations in the South China Sea (SCS) by analysis of crenarchaeal 16S rRNA gene, ammonia monooxygenase gene amoA involved in ammonia oxidation, and biotin carboxylase gene accA putatively involved in archaeal CO₂ fixation. Quantitative PCR analyses indicated that crenarchaeal amoA and accA gene abundances varied similarly with archaeal and crenarchaeal 16S rRNA gene abundances at all stations, except that crenarchaeal accA genes were almost absent in the epipelagic zone. Ratios of the crenarchaeal amoA gene to 16S rRNA gene abundances decreased ~2.6 times from the epi- to bathypelagic zones, whereas the ratios of crenarchaeal accA gene to marine group I crenarchaeal 16S rRNA gene or to crenarchaeal amoA gene abundances increased with depth, suggesting that the metabolism of Crenarchaeota may change from the epi- to meso- or bathypelagic zones. Denaturing gradient gel electrophoresis profiling of the 16S rRNA genes revealed depth partitioning in archaeal community structures. Clone libraries of crenarchaeal amoA and accA genes showed two clusters: the “shallow” cluster was exclusively derived from epipelagic water and the “deep” cluster was from meso- and/or bathypelagic waters, suggesting that niche partitioning may take place between the shallow and deep marine Crenarchaeota. Overall, our results show strong depth partitioning of crenarchaeal populations in the SCS and suggest a shift in their community structure and ecological function with increasing depth.     

Crenarchaeota and their role in the nitrogen cycle in a subsurface radioactive thermal spring in the Austrian Central Alps

Previous results from a 16S rRNA gene library analysis showed high diversity within the prokaryotic community of a subterranean radioactive thermal spring, the "Franz-Josef-Quelle" (FJQ) in Bad Gastein, Austria, as well as evidence for ammonia oxidation by crenarchaeota. This study reports further characterization of the community by denaturing gradient gel electrophoresis (DGGE) analysis, fluorescence in situ hybridization (FISH), and semiquantitative nitrification measurements. DGGE bands from three types of samples (filtered water, biofilms on glass slides, and naturally grown biofilms), including samples collected at two distinct times (January 2005 and July 2006), were analyzed. The archaeal community consisted mainly of Crenarchaeota of the soil-subsurface-freshwater group (group 1.1b) and showed a higher diversity than in the previous 16S rRNA gene library analysis, as was also found for crenarchaeal amoA genes. No bacterial amoA genes were detected. FISH analysis of biofilms indicated the presence of archaeal cells with an abundance of 5.3% (+/-4.5%) in the total 4',6-diamidino-2-phenylindole (DAPI)-stained community. Microcosm experiments of several weeks in duration showed a decline of ammonium that correlated with an increase of nitrite, the presence of crenarchaeal amoA genes, and the absence of bacterial amoA genes. The data suggested that only ammonia-oxidizing archaea (AOA) perform the first step of nitrification in this 45 degrees C environment. The crenarchaeal amoA gene sequences grouped within a novel cluster of amoA sequences from the database, originating from geothermally influenced environments, for which we propose the designation "thermal spring" cluster and which may be older than most AOA from soils on earth.     

Abundance, diversity, and activity of ammonia-oxidizing prokaryotes in the coastal Arctic ocean in summer and winter

Ammonia oxidation, the first step in nitrification, is performed by certain Beta- and Gammaproteobacteria and Crenarchaea to generate metabolic energy. Ammonia monooxygenase (amoA) genes from both Bacteria and Crenarchaea have been found in a variety of marine ecosystems, but the relative importance of Bacteria versus Crenarchaea in ammonia oxidation is unresolved, and seasonal comparisons are rare. In this study, we compared the abundance of betaproteobacterial and crenarchaeal amoA genes in the coastal Arctic Ocean during summer and winter over 2 years. Summer and winter betaproteobacterial amoA clone libraries were significantly different, although the gene sequences were similar to those found in temperate and polar environments. Betaproteobacterial and crenarchaeal amoA genes were 30- to 115-fold more abundant during the winter than during the summer in both years of the study. Archaeal amoA genes were more abundant than betaproteobacterial amoA genes in the first year, but betaproteobacterial amoA was more abundant than archaeal amoA the following year. The ratio of archaeal amoA gene copies to marine group I crenarchaeal 16S rRNA genes averaged 2.9 over both seasons and years, suggesting that ammonia oxidation was common in Crenarchaea at this location. Potential nitrification rates, as well as the total amoA gene abundance, were highest in the winter when competition with phytoplankton was minimal and ammonium concentrations were the highest. These results suggest that ammonium concentrations were important in determining the rates of ammonia oxidation and the abundance of ammonia-oxidizing Betaproteobacteria and Crenarchaea.     

Vertical structure of archaeal communities and the distribution of ammonia monooxygenase A gene variants in two meromictic High Arctic lakes

The distribution of archaeal amoA and 16S rRNA genes was evaluated in two marine-derived, meromictic lakes in the Canadian High Arctic: Lake A and Lake C1 on the northern coast of Ellesmere Island. The amoA gene was recorded in both lakes, with highest copy numbers in the oxycline. Sequence analysis showed that amoA from the two lakes shared 94% similarity, indicating at least two phylogenetically distinct clusters. Clone libraries of archaeal 16S rRNA genes from Lake A revealed strong vertical differences in archaeal community diversity and composition down the water column. The oxic layer was dominated by one group of Euryarchaeota affiliated to the Lake Dagow Sediment (LDS) cluster. This group was absent from the oxycline, which had an extremely low archaeal diversity of two phylotypes. Both belonged to the Crenarchaeota Marine Group I (MGI), the marine group that has been linked to archaeal amoA ; however, there was a low ratio of amoA to MGI copy numbers, suggesting that many MGI Archaea did not carry the amoA gene. The anoxic zone contained representatives of the RC-V (Rice Cluster-V) and LDS clusters of Euryarchaeota. These results show the strong vertical differentiation of archaeal communities in polar meromictic lakes, and they suggest archaeal nitrification within the oxycline of these highly stratified waters.     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes

The annual changes in the composition and abundance of ammonia-oxidizing archaea (AOA) were analyzed monthly in surface waters of three high mountain lakes within the Limnological Observatory of the Pyrenees (LOOP; northeast Spain) using both 16S rRNA and functional (ammonia monooxygenase gene, amoA) gene sequencing as well as quantitative PCR amplification. The set of biological data was related to changes in nitrogen species and to other relevant environmental variables. The whole archaeal assemblage was dominated by phylotypes closely related to the crenarchaeal 1.1a group (58% ± 18% of total 16S rRNA gene sequences), and consistent structural changes were detected during the study. Water temperature was the environmental variable that better explained spring, summer, and winter (ice-covered lakes) archaeal assemblage structure. The amoA gene was detected year round, and seasonal changes in amoA gene composition were well correlated with changes in the archaeal 16S rRNA gene pool. In addition, copy numbers of both the specific 1.1a group 16 rRNA and archaeal amoA genes were well correlated, suggesting that most freshwater 1.1a Crenarchaeota had the potential to carry out ammonia oxidation. Seasonal changes in the diversity and abundance of AOA (i.e., amoA) were better explained by temporal changes in ammonium, the substrate for nitrification, and mostly nitrite, the product of ammonia oxidation. Lacustrine amoA gene sequences grouped in coherent freshwater phylogenetic clusters, suggesting that freshwater habitats harbor typical amoA-containing ecotypes, which is different from soils and seas. We observed within the freshwater amoA gene sequence pool a high genetic divergence (translating to up to 32% amino acid divergence) between the spring and the remaining AOA assemblages. This suggests that different AOA ecotypes are adapted to different temporal ecological niches in these lakes.     

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change.     

Comparative analysis of 16S rRNA and amoA genes from archaea selected with organic and inorganic amendments in enrichment culture

We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the "root" clade, we detected no corresponding amoA gene. The amoA-containing archaea were present in media with either organic or inorganic amendments, whereas archaea representing the root clade were present only when organic amendment was used. Analysis of amoA gene abundance and expression, together with nitrification-coupled growth assays, indicated potential growth by autotrophic ammonia oxidation for members of two group 1.1b clades. Increased abundance of one of these clades, however, also occurred upon the addition of organic amendment. Finally, although amoA-containing group 1.1a archaea were present in enrichments, we detected neither expression of amoA genes nor evidence for nitrification-coupled growth of these organisms. These data support a model of a diverse metabolic community in mesophilic soil archaea that is just beginning to be characterized.     

Comparative analysis of archaeal 16S rRNA and <Emphasis Type="Italic">amoA</Emphasis> genes to estimate the abundance and diversity of ammonia-oxidizing archaea in marine sediments

Considering their abundance and broad distribution, non-extremophilic Crenarchaeota are likely to play important roles in global organic and inorganic matter cycles. The diversity and abundance of archaeal 16S rRNA and putative ammonia monooxygenase alpha-subunit (amoA) genes were comparatively analyzed to study genetic potential for nitrification of ammonia-oxidizing archaea (AOA) in the surface layers (0-1 cm) of four marine sediments of the East Sea, Korea. After analysis of a 16S rRNA gene clone library, we found various archaeal groups that include the crenarchaeotal group (CG) I.1a (54.8%) and CG I.1b (5.8%), both of which are known to harbor ammonia oxidizers. Notably, the 16S rRNA gene of CG I.1b has only previously been observed in terrestrial environments. The 16S rRNA gene sequence data revealed a distinct difference in archaeal community among sites of marine sediments. Most of the obtained amoA sequences were not closely related to those of the clones retrieved from estuarine sediments and marine water columns. Furthermore, clades of unique amoA sequences were likely to cluster according to sampling sites. Using real-time PCR, quantitative analysis of amoA copy numbers showed that the copy numbers of archaeal amoA ranged from 1.1 x 10(7) to 4.9 x 10(7) per gram of sediment and were more numerous than those of bacterial amoA, with ratios ranging from 11 to 28. In conclusion, diverse CG I.1a and CG I.1b AOA inhabit surface layers of marine sediments and AOA, and especially, CG I.1a are more numerous than other ammonia-oxidizing bacteria.     

Quantitative distribution of presumptive archaeal and bacterial nitrifiers in Monterey Bay and the North Pacific Subtropical Gyre

The recent isolation of the ammonia-oxidizing crenarchaeon Nitrosopumilus maritimus has expanded the known phylogenetic distribution of nitrifying phenotypes beyond the domain Bacteria. To further characterize nitrification in the marine environment and explore the potential crenarchaeal contribution to this process, we quantified putative nitrifying genes and phylotypes in picoplankton genomic libraries and environmental DNA samples from coastal and open ocean habitats. Betaproteobacteria ammonia monooxygenase subunit A (amoA) gene copy numbers were low or undetectable, in stark contrast to crenarchaeal amoA-like genes that were broadly distributed and reached up to 6 x 10(4) copies ml(-1). Unexpectedly, in the North Pacific Subtropical Gyre, a deeply branching crenarchaeal group related to a hot spring clade (pSL12) was at times abundant below the euphotic zone. Quantitative data suggested that the pSL12 relatives also contain archaeal amoA-like genes. In both coastal and open ocean habitats, close relatives of known nitrite-oxidizing Nitrospina species were well represented in genomic DNA libraries and quantitative PCR profiles. Planktonic Nitrospina depth distributions correlated with those of Crenarchaea. Overall, the data suggest that amoA-containing Crenarchaea are more phylogenetically diverse than previously reported. Additionally, distributional patterns of planktonic Crenarchaea and Nitrospina species suggest potential metabolic interactions between these groups in the ocean's water column.     

Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary

Submarine groundwater discharge to coastal waters can be a significant source of both contaminants and biologically limiting nutrients. Nitrogen cycling across steep gradients in salinity, oxygen and dissolved inorganic nitrogen in sandy 'subterranean estuaries' controls both the amount and form of nitrogen discharged to the coastal ocean. We determined the effect of these gradients on betaproteobacterial ammonia-oxidizing bacteria (β-AOB) and ammonia-oxidizing archaea (AOA) in a subterranean estuary using the functional gene encoding ammonia monooxygenase subunit A ( amoA ). The abundance of β-AOB was dramatically lower in the freshwater stations compared with saline stations, while AOA abundance remained nearly constant across the study site. This differing response to salinity altered the ratio of β-AOB to AOA such that bacterial amoA was 30 times more abundant than crenarchaeal amoA at the oxic marine station, but nearly 10 times less abundant at the low-oxygen fresh and brackish stations. As the location of the brackish mixing zone within the aquifer shifted from landward in winter to oceanward in summer, the location of the transition from a β-AOB-dominated to an AOA-dominated community also shifted, demonstrating the intimate link between microbial communities and coastal hydrology. Analysis of ammonia-oxidizing enrichment cultures at a range of salinities revealed that AOA persisted solely in the freshwater enrichments where they actively express amoA . Diversity (as measured by total richness) of crenarchaeal amoA was high at all stations and time points, in sharp contrast to betaproteobacterial amoA for which only two sequence types were found. These results offer new insights into the ecology of AOA and β-AOB by elucidating conditions that may favour the numerical dominance of β-AOB over AOA in coastal sediments.     

The phylogenetic affiliation of an uncultured population of ammonia-oxidizing bacteria harboring environmental sequences of amoA cluster-3

We investigated the phylogenetic diversity of ammonia-oxidizing bacteria (AOB) in Yellow Sea continental shelf sediment by the cloning and sequencing of PCR-amplified amoA and 16S rRNA genes. Phylogenetic analysis of the amoA-related clones revealed that the diversity of AOB was extremely low at the study site. The majority (92.7%) of amoA clones obtained belonged to a single cluster, environmental amoA cluster-3, the taxonomic position of which was previously unknown. Phylogenetic analysis on AOB-specific 16S rRNA gene sequences also demonstrated a very low diversity. All of the cloned 16S rRNA gene sequences comprised a single phylotype that belonged to the members of uncultured Nitrosospira cluster-1, suggesting that AOB belonging to the uncultured Nitrosospira cluster- 1 could carry amoA sequences of environmental amoA cluster-3.     

Changes in N-transforming archaea and bacteria in soil during the establishment of bioenergy crops

Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the landscape, which are mainly carried out by soil microbes. Here we explore the impact of four potential bioenergy feedstock crops (maize, switchgrass, Miscanthus X giganteus, and mixed tallgrass prairie) on nitrogen cycling microorganisms in the soil by monitoring the changes in the quantity (real-time PCR) and diversity (barcoded pyrosequencing) of key functional genes (nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The quantities of these N-cycling genes were relatively stable in all four crops, except maize (the only fertilized crop), in which the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia oxidizers. Deep sequencing revealed high diversity of nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA genes, with 229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in maize markedly decreased in the second year. Ordination analysis of T-RFLP and pyrosequencing results showed that the N-transforming microbial community structures in the soil under these crops gradually differentiated. Thus far, our two-year study has shown that specific N-transforming microbial communities develop in the soil in response to planting different bioenergy crops, and each functional group responded in a different way. Our results also suggest that cultivation of maize with N-fertilization increases the abundance of AOB and denitrifiers, reduces the diversity of AOA, and results in significant changes in the structure of denitrification community.     

Diversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River (China)

Here we report on the biodiversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in sediment samples from the Xinyi River, Jinagsu Province (China). The biodiversity of aerobic ammonium-oxidizing bacteria in the sediment was assessed using the amoA gene as functional marker. The retrieved amoA clones were affiliated to environmental sequences from freshwater habitats. The closest cultivated relative was Nitrosomonas urea. Anaerobic ammonium-oxidizing (anammox) bacteria were studied using anammox and planctomycete-specific 16S rRNA gene primers. The sediments contained 16S rRNA genes and bacterial cells closely related to the known anammox bacterium Candidatus'Brocadia anammoxidans'. Anaerobic continuous flow reactors were set up to enrich anammox organisms from the sediments. After an adaptation period of about 25 days the reactors started to consume ammonium and nitrite, indicating that the anammox reaction was occurring with a rate of 41-58 nmol cm(-3) h(-1). Community analysis of the enrichments by quantitative fluorescence in situ hybridization showed an increase in the abundance of anammox bacteria from < 1% to 6 +/- 2% of the total population. Analysis of the 16S rRNA genes showed that the enriched anammox organisms were related to the Candidatus'Scalindua' genus.     

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane

Methanotroph abundance was analyzed in control and long-term nitrogen-amended pine and hardwood soils using rRNA-targeted quantitative hybridization. Family-specific 16S rRNA and pmoA/amoA genes were analyzed via PCR-directed assays to elucidate methanotrophic bacteria inhabiting soils undergoing atmospheric methane consumption. Quantitative hybridizations suggested methanotrophs related to the family Methylocystaceae were one order of magnitude more abundant than Methyloccocaceae and more sensitive to nitrogen-addition in pine soils. 16S rRNA gene phylotypes related to known Methylocystaceae and acidophilic methanotrophs and pmoA/amoA gene sequences, including three related to the upland soil cluster Alphaproteobacteria (USCalpha) group, were detected across different treatments and soil depths. Our results suggest that methanotrophic members of the Methylocystaceae and Beijerinckiaceae may be the candidates for soil atmospheric methane consumption.     

Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.