Ammonia Oxidation Coupled to CO2 Fixation by Archaea and Bacteria In

Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil

Jennifer Pratscher, Marc G. Dumont, and Ralf Conrad1

Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany

Edited by James M. Tiedje, Center for Microbial Ecology, East Lansing, MI, and approved February 1, 2011 (received for review August 2, 2010)

Ammonia oxidation is an essential part of the global nitrogen fixation, one should be able to identify the active autotrophic cycling and was long thought to be driven only by bacteria. Recent ammonia-oxidizing prokaryotes using SIP. So far, DNA-SIP findings expanded this pathway also to the archaea. However, analyses successfully showed autotrophy of ammonia-oxidizing most questions concerning the metabolism of ammonia-oxidizing bacteria in sediments of a lake (14) and an estuary (15) but failed to detect CO fixation of ammonia-oxidizing archaea in agri- archaea, such as ammonia oxidation and potential CO2 fixation, 2 remain open, especially for terrestrial environments. Here, we in- cultural soil (16), although potential activity of these archaea in vestigated the activity of ammonia-oxidizing archaea and bacteria soil has been reported before (17, 18). DNA-SIP of grassland soil in an agricultural soil by comparison of RNA- and DNA-stable iso- revealed autotrophic ammonia oxidation of archaea (19), but tope probing (SIP). RNA-SIP demonstrated a highly dynamic and exactly which groups of archaeal ammonia oxidizers contributed to this process remains unclear. Furthermore, the efficiency of diverse community involved in CO fixation and carbon assimila- 2 DNA-SIP depends solely on replication of cells, thus excluding tion coupled to ammonia oxidation. DNA-SIP showed growth of microorganisms that might be active but not growing. In this the ammonia-oxidizing bacteria but not of archaea. Furthermore, case, RNA-SIP (20) is assumed to yield more detailed in- the analysis of labeled RNA found transcripts of the archaeal formation regarding activity. acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed The aim of this study was to investigate CO2 assimilation and labeled. These findings strongly suggest that ammonia- linked to nitrification of ammonia-oxidizing prokaryotes in an fi oxidizing archaeal groups in soil autotrophically xCO2 using the agricultural soil using RNA-SIP and DNA-SIP in parallel. We 3-hydroxypropionate–4-hydroxybutyrate cycle, one of the two also wanted to detect expression of archaeal amoA by mRNA fi fi Crenarchaeota pathways recently identi ed for CO2 xation in . Cat- catalyzed reporter deposition (CARD)-FISH. Our findings pro- alyzed reporter deposition (CARD)-FISH targeting the gene encod- vide further evidence that the contribution of nitrifying archaea amoA ing subunit A of ammonia monooxygenase ( ) mRNA and 16S to ammonia oxidation and CO2 fixation in terrestrial environ- rRNA of archaea also revealed ammonia-oxidizing archaea to be ments might be substantial. numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxi- Results

dizing archaea in soil to nitrification and CO2 assimilation and that Nitrification Activity in SIP Incubations. For SIP, agricultural soil 13 their importance to the overall archaeal community might be larger microcosms were incubated with 5% C-labeled CO2 or un- 12 than previously thought. labeled C-CO2 for 12 wk. Concentrations of 1–5% CO2 are considered typical in soil (21). Weekly fertilization of the soil μ μ · −1 mmonia oxidation, the first step in nitrification, is crucial for with either 15 g or 100 g (NH4)2SO4-N g dryweight of soil Athe global nitrogen cycle. For a long time bacteria were (d.w.s.) resulted in stepwise production and increase of nitrate believed to be solely responsible for this process and exclusively (Fig. S1), whereas nitrate concentration in the unfertilized con- to possess the genes for the ammonia monooxygenase (amo), the trol did not increase. As expected, the largest nitrate production fi was observed in the microcosms fertilized with the higher con- key enzyme of nitri cation (1). Now there is increasing evidence μ · −1 that archaea also are involved. Archaeal genes encoding subunit centration of ammonia (100 gNg d.w.s.). Ammonium and A of ammonia monooxygenase (amoA) have been found to oc- nitrite did not accumulate over time, indicating that nitrate cur in a wide variety of environments including marine systems, production indeed resulted from ammonia oxidation. Ammonia – and nitrate concentrations were not balanced because net nitri- hot springs, and soils (2 5). Furthermore, molecular studies fi fi revealed that ammonia-oxidizing archaea often outnumber the cation generally underestimates gross nitri cation in soils be- nitrifying bacteria in most environments by orders of magnitude cause of additional nitrogen cycling (22). (3, 5, 6). These findings all demonstrate the potentially signifi- cant role of archaea in the process of nitrification. Evidence also RNA-SIP. For RNA-SIP of ammonia-oxidizing prokaryotes, buoyant density centrifugation was conducted with all RNA ex- suggests the assignment of the ammonia-oxidizing archaea 12 13 should be assigned to the archaeal phylum Thaumarchaeota in- tracts from C and C microcosms after 8 and 12 wk of in- stead of to the Crenarchaeota (7, 8). In addition, these archaea cubation. The quantitative distribution of archaeal and bacterial may be of importance for the global carbon cycle. The ammonia- amoA transcripts in these gradients was analyzed by quantitative oxidizing archaea isolated from aquatic environments were all PCR (qPCR) of cDNA (Fig. 1). The copy numbers obtained – represent mean results from the triplicate microcosms and re- shown to be autotrophs (9 11), like their bacterial counterparts, 13 and analysis of 13C-bicarbonate–labeled lipid biomarkers of peated qPCR analyses. After 8 wk of incubation with 5% CO2 natural Crenarchaeota in the North Sea indicated an autotrophic metabolism (12). However, it still is unclear whether ammonia- oxidizing archaea in soil also have an autotrophic metabolism Author contributions: J.P., M.G.D., and R.C. designed research; J.P. performed research; and to what extent they are functionally active. An answer to this J.P. analyzed data; and J.P. and R.C. wrote the paper. question and a link of phylogeny to function could be provided The authors declare no conflict of interest. by stable isotope probing (SIP) of nucleic acids. This technique This article is a PNAS Direct Submission. allows the specific identification of microorganisms assimilating Data deposition: The sequences reported in this paper have been deposited in the Gen- labeled substances, most commonly carbon from a particular Bank database (accession nos. HM996921–HM996934, HQ293120–HQ293148,and 13C-labeled substrate (13). Direct demonstration of ammonia HQ685759–HQ685837). oxidation by this method is not possible, because nitrite, the 1To whom correspondence should be addressed. E-mail: [email protected]. product of ammonia oxidation, is not assimilated. However, as- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suming that ammonia oxidation is coupled to autotrophic CO2 1073/pnas.1010981108/-/DCSupplemental.

4170–4175 | PNAS | March 8, 2011 | vol. 108 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1010981108 Downloaded by guest on September 24, 2021 −1 12 and fertilization with 15 μgN·g d.w.s., the copy number of observed. Also, controls with CO2 and the unfertilized samples archaeal amoA transcripts already showed detectable labeling in did not show any labeling (Fig. S3). These results suggest that, − the heavy fraction (1.81–1.83 g·mL 1) (Fig. 1A). This clear la- although archaea were actively involved in ammonia oxidation beling could not be seen with the fertilization treatment with 100 and CO assimilation, as shown in the RNA-SIP approach, they − 2 μgN·g 1 d.w.s. (Fig. 1C). Instead, a shift of the archaeal amoA did not replicate. By contrast, the ammonia-oxidizing bacteria mRNA toward the partially labeled, intermediate gradient acquired heavy DNA to such an extent that those not pro- − fraction (1.79–1.80 g·mL 1)of13C gradients was observed. This liferating could no longer be detected in the light DNA fractions. result indicates that archaea might have been inhibited by the This notion was supported by a comparison of the copy numbers − elevated ammonia concentration in the 100 μgN·g 1 d.w.s. of archaeal and bacterial amoA genes in the initial soil versus treatment, resulting in a slower activation and lower activity of copy numbers in the incubated soil, determined by qPCR. Al- ammonia oxidation. This notion is supported by the results of 12- though ammonia-oxidizing bacteria showed strong growth within − wk incubation. Although the archaeal amoA transcripts in the 15 the 12 wk of incubation (copy number·g 1 d.w.s. : 5.47 ± 0.75 × − μgN·g 1 d.w.s. treatment were shifted almost completely into the 106 in initial soil and 7.80 ± 0.67 × 107 after 12-wk incubation), this − heavy fraction (Fig. 1B), the transcripts of the 100 μgN·g 1 d.w.s. growth could not be observed for the archaea containing amoA − fertilization were still labeled only partially (Fig. 1D). The copy (copy number·g 1 d.w.s. : 4.77 ± 0.51 × 107 in initial soil and 5.35 ± number of bacterial amoA transcripts also peaked after 8 wk of 0.39 × 107 after 12-wk incubation). 13 incubation in the fractions with heavy RNA from the CO2 treatment (Fig. 1 E and G). Here, the labeling was stronger with Phylogenetic Analyses of Archaeal 16S rRNA in RNA-SIP and of amoA − the 100 μgN·g 1 d.w.s. treatment (Fig. 1E) than in the micro- Transcripts in RNA- and DNA-SIP. The heavy and light RNA frac- − − cosms fertilized with 15 μgN·g 1 d.w.s. (Fig. 1G). After 12 wk, tions (1.815 and 1.783 g·mL 1, respectively) from microorgan- bacterial amoA mRNA in both treatments was shifted com- isms incubated with 13CO and treated with low fertilizer levels − 2 pletely to the heavy fraction (Fig. 1 F and H). No labeling was (15 μgN·g 1 d.w.s.) were used after 8 wk of incubation to analyze 12 observed in the unfertilized treatment or the controls with CO2 the sequences of archaeal 16S rRNA transcripts. Sequence (Fig. S2), demonstrating that the heavy RNA from the other analysis of 16S rRNA revealed that all sequences (40 clones), gradients indeed resulted from true label incorporation into se- from both light (20 clones) and heavy fractions (20 clones), lected microbes in SIP (23, 24). The fact that no labeling was showed highest similarity (95% maximum identity) to the am- observed in the unfertilized treatment also shows that CO2 fix- monia oxidizer Nitrososphaera gargensis (Fig. S4). Light and ation was coupled to ammonia oxidation. heavy RNA fractions and light DNA fractions from the micro- organisms incubated with 13CO and treated with low fertilizer − 2 DNA SIP. Gradient centrifugation of DNA was performed with levels (15 μgN·g 1 d.w.s.) for 8 or 12 wk also were used to an- 12 13 all respective DNA extracts from CO2- and CO2-exposed alyze archaeal amoA transcripts and genes, respectively. Analysis microcosms after 8 and 12 wk of incubation. The quantitative of these sequences showed explicit differences among the gra- distribution of archaeal and bacterial amoA genes in these gra- dient fractions regarding presence and activity of different am- dients was analyzed by qPCR (Fig. 2). The obtained copy num- monia-oxidizing archaeal groups (Fig. 3A). Although after 8 and bers represent mean results from the triplicate microcosms and 12 wk of incubation DNA fractions were dominated completely repeated qPCR analyses. After 8 wk of incubation, the copy by a particular cluster of sequences (cluster 1), this abundance numbers of archaeal and bacterial amoA genes for both fertil- was not reflected in the transcripts, suggesting these archaea ization treatments peaked only in the light fractions (1.69–1.72 exhibited only minor activity, probably increasing after 12 wk − g·mL 1) (Fig. 2 A, C, E, and G). Note that the small shift in as shown by the higher abundance in the light RNA fraction. buoyant density results from different GC contents of archaea Sequences of an additional cluster (cluster 3) also appeared only and bacteria (25, 26). Although no labeling was detected for in the light fractions, indicating potential ammonia-oxidizing ac- archaea after 12 wk of incubation (Fig. 2 B and D), the bacterial tivity but no assimilation of labeled carbon. However, after 8 wk of amoA genes were shifted completely to the heavy fraction (1.73– incubation, the heavy RNA fraction consisted exclusively of −1 1.76 g·mL ) (Fig. 2 F and H). A difference regarding the N a specific cluster (cluster 2) demonstrating active CO2 fixation and fertilization treatments, as seen in the RNA-SIP, could not be incorporation of labeled carbon. After 12 wk of incubation, the

Fig. 1. Distribution of amoA transcripts from archaea (A–D) and bacteria (E–H)in RNA-SIP gradients after incubation for 8 wk (A, C, E, and G) or 12 wk(B, D, F, and H) with 13 CO2 and fertilization with 15 μg(A, B, E, and F) or 100 μg(C, D, G, and H)(NH4)2SO4- − N·g 1 d.w.s. Distribution of amoA tran-

scripts was measured by qPCR of cDNA from MICROBIOLOGY gradient fractions.

Pratscher et al. PNAS | March 8, 2011 | vol. 108 | no. 10 | 4171 Downloaded by guest on September 24, 2021 Fig. 2. Distribution of amoA genes from archaea (A–D) and bacteria (E–H) in DNA-SIP gradients after incubation for 8 wk (A, C, E, 13 and G) and 12 wk (B, D, F, and H) with CO2 and fertilization with 15 μg(A, B, E,andF)or −1 100 μg(C, D, G,andH)(NH4)2SO4-N·g d.w.s. Distribution of amoA gene abundance was measured by qPCR of DNA from gradient fractions.

heavy RNA fraction also showed only one amoA cluster (cluster fraction and consisted exclusively of a cluster of archaeal accA 4), indicating that these microorganisms were activated more sequences (cluster RH_HF), most closely related to Nitro- slowly but took over the incorporation of label either by CO2 sopumilus maritimus, Crenarchaeum symbiosum, and accA genes fixation or general carbon assimilation. An extended tree is shown of marine Crenarchaeota from deep Tyrrhenian Sea (27). These in Fig. S5. results strongly indicate an involvement of the acetyl-CoA/ propionyl-CoA carboxylase (accA/pccB) in the CO2-fixation Archaeal Acetyl-CoA Carboxylase Alpha Subunit Transcripts Detected process of ammonia-oxidizing archaea in soil. in Heavy RNA Fractions. The labeled and unlabeled RNA fractions − (1.815 and 1.783 g·mL 1, respectively) from the microorganisms mRNA CARD-FISH of Archaeal amoA and Abundance of Ammonia- 13 incubated with CO2 and treated with low fertilizer levels (15 μg Oxidizing Archaea and Bacteria In Soil. To investigate visually the − N·g 1 d.w.s.) were used after 12 wk of incubation to generate expression of amoA mRNA in ammonia-oxidizing archaea, clone libraries targeting bacterial and archaeal acetyl-CoA car- CARD-FISH of amoA transcripts and archaeal 16S rRNA was boxylase alpha subunit (accA) transcripts (Fig. 3B). AccA conducted, based on the protocol by Pernthaler and Amann (28). sequences could be detected only in the library of the heavy Clones and soil samples were hybridized with an archaeal amoA

Fig. 3. Phylogenetic affiliation of putative amoA (A) and accA (B) sequences derived from SIP gradient fractions of soil after incubation and fertilization with −1 13 15 μgN·g d.w.s. (A) amoA transcript and gene clones from CO2 RNA- and DNA-SIP gradient fractions of soil after 8 and 12 wk of incubation. The amoA clones are shown as clusters 1–4 (GenBank accession nos. HQ685759–HQ685837), and relative abundances of respective cluster sequences in the clone libraries of the different RNA- and DNA-SIP gradient fractions are included as bar charts with percentages. The tree is rooted with the amoA gene of Nitrosospira 13 briensis (U76553). (B) Putative AccA/PccB transcript clones derived from CO2 RNA-SIP gradient fractions of soil after 12 wk of incubation. The accA clones from 13C-labeled heavy RNA are shown as cluster RH_HF (GenBank accession nos. HM996921–HM996934). The tree is rooted with accA gene of Haloqua- dratum walsbyi DSM 16790 (YP_658717). Neighbor-joining analysis using 1,000 bootstrap replicates was used to infer tree topology, and the nodes with the percentage of bootstrap resampling above 90% are indicated by filled circles. (Scale bars: 10% amino acid sequence divergence.)

4172 | www.pnas.org/cgi/doi/10.1073/pnas.1010981108 Pratscher et al. Downloaded by guest on September 24, 2021 mRNA antisense probe, followed by detection with an anti-DIG consistently were detected solely in the unlabeled fractions antibody labeled with HRP and signal amplification by catalyzed throughout the whole incubation period (probably suggesting reporter deposition with fluorescein-labeled tyramide. For soil heterotrophic or mixotrophic activity), heavy RNA fractions samples, detection of mRNA also was coupled to CARD-FISH harbored one specific cluster exclusively after 8 and 12 wk of of archaeal 16S rRNA using the probe Arch915. The specificity incubation, respectively. Because incorporation of 13C by cluster of the amoA mRNA antisense probe for ammonia-oxidizing ar- 2 and bacterial amoA occurred simultaneously, at 8 wk of in- chaea in this soil was tested with expression clones (Fig. S6). cubation, the possibility that this cluster was labeled by cross- Induced cells showed strong hybridization signals (Fig. S6 A and feeding can be excluded, indicating that these ammonia-oxidizing B), but no signal was observed in uninduced cells (Fig. S7)orin archaea indeed fixed CO2 autotrophically. Exclusive labeling and clones expressing the partial amoA gene of ammonia-oxidizing detection of specific amoA transcripts after 12 wk of incubation bacteria (Fig. S6 C and D). These results indicate a high speci- could be explained either by autotrophic CO2 fixation or by spe- ficity of the amoA mRNA CARD-FISH. The hybridizations of cific cross-feeding on labeled carbon compounds derived from microorganisms in the soil incubated for 12 wk with 5% CO2 and ammonia-oxidizing bacteria. μ · −1 + fertilized weekly with 15 gNg d.w.s. resulted in good signals Our results show that NH4 fertilization stimulated carbon for both the amoA and 16S rRNA CARD-FISH (Fig. 4). Only assimilation by archaeal ammonia oxidizers, although CO2 fixa- cells that exhibited a signal with the 16S rRNA probe Arch915 tion and ammonia oxidation were stimulated to a greater extent − (Fig. 4 B and E) also showed detection by amoA mRNA FISH by the lower concentration of ammonia (15 μgN·g 1 d.w.s.). The − (Fig. 4 A and D). Half of the detected archaeal cells in this in- 100 μgN·g 1 d.w.s. treatment led to incomplete labeling of ar- cubated soil also showed a signal for amoA expression. To test chaeal amoA mRNA even after 12 wk of incubation. This ob- this observation further, the copy numbers of amoA genes and servation strengthens the hypothesis that ammonia-oxidizing 16S rRNA genes in the incubated soil, as determined by qPCR, archaea are adapted to low-nutrient environments and are were compared. Although ammonia-oxidizing bacteria slightly inhibited by high-level fertilization (17, 29–31). Our results agree outnumbered their archaeal counterparts, most likely because of with previous findings in Nitrososphaera gargensis isolated from the proliferation also observed in DNA-SIP, and made up 4% of a hot spring, which also was inhibited by relatively high con- − the bacterial community (copy number·g 1 d.w.s.: amoA, 7.80 ± centrations of ammonium (11), and a recent publication dem- 0.67 × 107; 16S rRNA: 2.02 ± 0.23 × 109)], archaea containing − onstrating growth of ammonia-oxidizing archaea in soil only at amoA accounted for 54% of all archaea (copy number·g 1 d.w.s.: acetylene-sensitive nitrification under low ammonia concen- amoA, 5.35 ± 0.39 × 107; 16S rRNA, 9.83 ± 0.91 × 107), sup- trations (18). Like Jia and Conrad (16), however, we were not porting the CARD-FISH observation. These results indicate that able to detect labeling of archaeal amoA when using DNA-SIP. ammonia-oxidizing archaea might play an important role in the Although RNA-SIP targeting amoA of ammonia-oxidizing ar- archaeal community in soil. chaea clearly demonstrated that labeled carbon was assimilated, DNA-SIP revealed no growth of these organisms, because de- Discussion tection of label incorporation in DNA-SIP can take place only Whether ammonia-oxidizing archaea in soil can assimilate CO2 when cells are actively replicating. We assume that the ammonia remains uncertain. A previous DNA-SIP–based study by Jia and concentrations in the soil for both fertilization treatments might Conrad (16) using this soil detected only labeling of bacteria by have allowed high and dynamic activity of ammonia-oxidizing CO2 fixation and concluded that ammonia-oxidizing archaea in archaea, as seen in RNA-SIP and phylogenetic analyses of amoA soil might be heterotrophic or mixotrophic rather than autotro- sequences, but still did not provide favorable conditions for them phic. We were able to demonstrate active CO2 fixation and to grow. This uncoupling generally should be considered when carbon assimilation coupled to ammonia oxidation by archaea in investigating the activity of microorganisms in natural environ- an agricultural soil using an RNA-SIP approach. Archaeal amoA ments, because methods depending on cell growth to detect in- transcripts were labeled consistently during incubation of soil corporation of label might not or only insufficiently detect cells that 13 microcosms with CO2 and fertilization with either 15 μg or 100 are active but grow more slowly. For these microorganisms, RNA- −1 μg (NH4)2SO4-N·g d.w.s. Controls without fertilization and SIP might be the more appropriate approach. 12 with CO2 did not show any labeling, confirming that CO2 fix- Unlike the archaea, the amoA copies of ammonia-oxidizing ation was coupled to ammonia oxidation and that labeling bacteria were labeled completely in RNA- and DNA-SIP for resulted from true label incorporation. Analyses of archaeal both levels of fertilization after 12 wk of incubation. This result amoA transcripts and genes from gradient fractions of the was not surprising, because it has been known for a long time −1 microcosms with the lower level of fertilization (15 μgN·g that ammonia-oxidizing bacteria fixCO2 using the Calvin cycle d.w.s.) revealed clear differences and dynamic changes of ar- and its key enzyme, the ribulose bisphosphate carboxylase chaeal ammonia oxidizers representing amoA clusters that differ (RubisCO) (32, 33). The SIP results demonstrated activity and in activity and in assimilation of carbon. Although some groups growth to such an extent that the whole community of ammonia-

Fig. 4. Detection of amoA mRNA transcripts by application of CARD-FISH with archaeal amoA antisense probe in agri-

cultural soil after 12 wk incubation with 5% CO2 and fer- tilization with 15 μgN·g−1 d.w.s. Fluorescence images for amoA CARD-FISH (A and D) and respective phase-contrast images (C and F) are shown. Archaeal cells in the soil in-

cubation also were detected by 16S rRNA CARD-FISH using MICROBIOLOGY the HRP-labeled probe Arch915 (B and E). (Scale bars:10 μm.)

Pratscher et al. PNAS | March 8, 2011 | vol. 108 | no. 10 | 4173 Downloaded by guest on September 24, 2021 oxidizing bacteria could be detected only in the heavy fractions. was air dried, sieved through 1-mm mesh, homogenized, and stored at 4 °C The labeling of amoA mRNA took place more rapidly in the until further use. Incubation for SIP with 5% CO2 was performed in triplicate − fertilization treatments with 100 μgN·g 1 d.w.s., showing that for each treatment. Soil (10 g d.w.s.) was incubated at 60% maximum water- holding capacity, 25 °C, in darkness, in 120-mL serum bottles capped with nitrifying bacteria, as expected, responded even better to higher 12 13 fi butyl stoppers. Five percent of CO2 ( CO2 or CO2) was added to the concentrations of ammonia. In addition, we were able speci - μ μ · −1 cally and quantitatively to detect archaeal cells expressing amoA headspace, and the soil was fertilized with 100 gor15 g(NH4)2SO4-N g in incubated soil directly by mRNA CARD-FISH using expression d.w.s., respectively, dissolved in distilled water. Nitrogen-free controls re- ceived an equal amount of distilled water. Every week, bottles were flushed clones as controls. The high abundance of ammonia-oxidizing with synthetic air (20% O , 80% N ), 5% of CO was added, and fertilization archaea (∼50%) within the archaea in this soil, as visualized by 2 2 2 fl fi treatments were renewed. For chemical analysis of pH, ammonium, nitrite, uorescence microscopy, also was con rmed by qPCR of amoA and nitrate, aliquots of the soil were removed from each treatment every and 16S rRNA genes. Because ammonia-oxidizing archaea such week (SI Materials and Methods). as Nitrosopumilus maritimus are thought to harbor only one copy of the 16S rRNA gene and only one copy of the amoA gene, the Nucleic Acid Extraction and SIP Fractionation. After 8 and 12 wk of incubation 13 12 gene copy numbers derived by qPCR should be equivalent to cell with CO2 and CO2, respectively, 0.5 g of soil from each bottle was sam- numbers (34, 35). We also analyzed the archaeal 16S rRNA pled, frozen immediately in liquid nitrogen, and stored at −80 °C until fur- transcripts from light and heavy fractions of RNA-SIP and ob- ther processing. Nucleic acids were extracted from soil using an SDS-based served a close relation to Nitrososphaera gargensis. Taken together, protocol (48) with minor modifications. Soil (0.5 g) was mixed with 200 μLof these results demonstrate the high relative abundance of ammo- zirconia-silica beads (0.1 mm; Roth) and 1 mL of SDS extraction buffer in 2.0- nia-oxidizing archaea among the overall archaeal community mL screw-cap tubes. Cells were lysed in a FastPrep beat-beating system for − in soil. Furthermore, we wanted to determine which pathway 45 s at 6 m·s 1, and supernatants were extracted using phenol chloroform isoamyl alcohol (25:24:1) and chloroform isoamyl alcohol (24:1). Nucleic acids enables ammonia-oxidizing archaea in soil to fixCO2. Two au- totrophic carbon-fixation cycles have been described recently in were precipitated with polyethylene glycol (PEG) 6000 solution (20%) and μ μ Crenarchaeota, the dicarboxylate–4-hydroxybutyrate cycle and dissolved in 100 L of nuclease-free water. For SIP of RNA, 50 L of extract – was treated with RNase-free DNase I for digestion of DNA. RNA was purified the 3-hydroxypropionate 4-hydroxybutyrate cycle, and all Cren- − archaeota studied so far use one cycle or the other (36). Because using the RNeasy Mini Kit (Qiagen) and stored at 80 °C until further use. – Integrity of nucleic acids was checked on agarose gels, and concentration of the oxygen sensitivity of some of its enzymes, the dicarboxylate was determined using a NanoDrop instrument (Thermo Fisher Scientific). SIP hydroxybutyrate cycle is restricted to anaerobic or microaerobic fractionation of total DNA extract (5.0 μg) was performed with an initial CsCl Crenarchaeota of the orders Thermoproteales and Desulfur- · −1 × – – buoyant density of 1.72 g mL subjected to centrifugation at 177,000 g for ococcales (37 39). The enzymes of the hydroxypropionate 36 h at 20 °C (25). Gradient centrifugation of RNA was carried out in cesium hydroxybutyrate cycle, on the other hand, are oxygen tolerant. trifluoroacetate (CsTFA) as described previously (25) with an initial buoyant − Therefore, this cycle fits well with the lifestyle of aerobic Cren- density of 1.79 g·mL 1 and centrifugation at 130,000 × g for 65 h at 20 °C. archaeota (40). The hydroxypropionate–hydroxybutyrate cycle DNA and RNA gradients were fractionated from bottom to top by displacing occurs in the autotrophical crenarchaeal order Sulfolobales, e.g., the gradient medium with nuclease-free water at the top of the tube using fi fl · −1 Metallosphaera sedula (41–43). The CO2-fixing enzyme of this a syringe pump (Kent Scienti c) at a ow rate of 0.45 mL min , generating process is the bifunctional biotin-dependent acetyl-CoA/propionyl- 12 fractions per density gradient. The density of each fraction was de- CoA carboxylase (Acc/Pcc). Recent studies using genome analysis termined by refractometry (Reichert). DNA was recovered by PEG 6000 pre- have detected sequences of the accA/pccB in members of the cipitation with glycogen (49) and dissolved in 30 μL of nuclease-free water. mesophilic marine group I Crenarchaeota, including ammonia RNA was precipitated from CsTFA with two volumes of ethanol and 20 μg μ oxidizers Crenarchaeum symbiosum and Nitrosopumilus spp. (36, glycogen and resuspended in 10 L of nuclease-free water. RNA samples 43, 44), and in metagenomic libraries of uncultured ammonia-ox- from density gradient fractions were reverse transcribed with random hex- idizing marine Crenarchaeotes (27, 45–47), but the existence of amer primers (Invitrogen) and M-MLV reverse transcriptase (Promega). these sequences could not be linked to functionality. A very recent amoA publication using DNA-SIP targeted another key gene of this qPCR of Genes. The abundance of archaeal amoA genes and transcripts in the different SIP fractions was quantified by qPCR using primers amo196F pathway, the hcd gene encoding the 4-hydroxybutyryl-CoA dehy- μ fi and amo277R as previously described (5, 16). The 25- L reaction mixture dratase, to demonstrate autotrophic CO2 xation by soil thau- contained 12.5 μL of SYBRGreen Jump-Start Taq ReadyMix, 0.5 μM of each marchaea but did not assign this activity to certain archaeal groups −1 primer, 200 ng BSA·mL , 4.0 mM MgCl2, and 1.0 μL template DNA or cDNA (19). Our results now show that transcripts of accA/pccB, closely (50). The abundance of bacterial amoA genes and transcripts in the different related to the sequences of the ammonia-oxidizing marine Cren- SIP fractions was quantified by qPCR using primers amoA-1F and amoA-2R as archaeota, not only were expressed but also were labeled by as- previously described (16, 51). The 25-μL reaction mixture contained 12.5 μL 13 similation of CO2.Thesefindings strongly suggest that specific of SYBRGreen Jump-Start Taq ReadyMix, 0.5 μM of each primer, 200 ng −1 groups of ammonia-oxidizing archaea in upland soils are able to fix BSA·mL , 3.0 mM MgCl2, and 1.0 μL template DNA or cDNA. All assays were CO2 autotrophically using the hydroxypropionate–hydrox- performed in an iCycler (Applied Biosystems), respective qPCR standards ybutyrate cycle, hence providing an additional sink for CO2 in were used, and controls always were run with water instead of DNA or cDNA terrestrial environments. extract. PCR efficiencies for all qPCR assays were between 90 and 104% with In summary, using mRNA-SIP, we were able to show that r2 values between 0.976 and 0.997. ammonia-oxidizing archaea were actively involved in microbial ammonia oxidation in an agricultural soil and exhibited a diverse Analysis of Archaeal 16S rRNA and accA Transcripts and Archaeal amoA Genes and Transcripts from Light and Heavy Fractions of SIP. cDNA and DNA (only for and dynamic activity in carbon assimilation. Although some 13 amoA) of the heavy and light fractions of the CO2 treatment with 15 μg groups probably demonstrated heterotrophic activity, different −1 (NH4)2SO4-N·g d.w.s. was used for PCR amplification of archaeal 16S rRNA highly active groups did fixCO2 autotrophically, presumably via the hydroxypropionate–hydroxybutyrate cycle. These results and and amoA cDNA and DNA fragments using primers Arch109F/Arch934R and Amo19f/Amo643r, respectively, as previously described (52, 16). Bacterial the observed numerical importance of the archaeal ammonia and archaeal accA cDNA fragments were amplified by primers PcB_388F and oxidizers in the overall archaeal community in this environment PcB_1271R (27). PCR products were cloned using pGEM-T Easy vector and give enhanced insights into the function and characteristics of Escherichia coli JM109-competent cells (Promega). Sequencing was carried ammonia-oxidizing archaea in soil. out on an ABI 3130 genetic analyzer (Applied Biosystems) and analyzed by DNAStar software package. Phylogenetic trees were reconstructed from Materials and Methods sequence data using the ARB software package (53). accA and amoA tree Soil Incubation. Soil was sampled in April 2009 using soil cores (40 cm long) topologies were checked by neighbor-joining algorithm using 1,000 boot- from maize plots at the long-term experiment field site of the University of strap replicates and were verified with trees calculated using maximum Giessen, Germany. Maize, wheat, and barley are rotated annually at the field likelihood. Archaeal 16S rRNA tree topology was checked by neighbor- site. The field site and soil properties were described previously (16). The soil joining algorithm using 500 bootstrap replicates and Jukes–Cantor correc-

4174 | www.pnas.org/cgi/doi/10.1073/pnas.1010981108 Pratscher et al. Downloaded by guest on September 24, 2021 tion of distances. The sequences reported in this paper have been deposited spectively, and were expressed using vector pBAD as previously described by – + in the GenBank database (accession nos. HM996921 HM996934 for accA, Pernthaler and Amann (28). After 12 wk of fertilization with 15 μgNH4 - − HQ685759–HQ685837 for amoA, and HQ293120–HQ293148 for archaeal N·g 1 d.w.s., clones and soil samples were fixed, and CARD-FISH was per- 16S rRNA). formed as described in SI Materials and Methods.

amoA CARD-FISH of mRNA and Archaeal 16S rRNA. To generate controls for ACKNOWLEDGMENTS. We are grateful to Dr. Lothar Behle-Schalk for the mRNA CARD-FISH, partial archaeal (positive control) and bacterial (negative access to the long-term experimental trial of the University of Giessen. We control) amoA genes from soil were cloned into E. coli Top10-competent thank Peter Claus, Melanie Klose, and Bianca Pommerenke for skillful cells using primers amo111F/amo643R (54) and amoA-1F/amoA-2R (55), re- technical assistance.

1. Kowalchuk GA, Stephen JR (2001) Ammonia-oxidizing bacteria: A model for 30. Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA (2009) molecular microbial ecology. Annu Rev Microbiol 55:485–529. Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and 2. Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and Bacteria. Nature 461:976–979. diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. 31. Di HJ, et al. (2010) Ammonia-oxidizing bacteria and archaea grow under contrasting Proc Natl Acad Sci USA 102:14683–14688. soil nitrogen conditions. FEMS Microbiol Ecol 72:386–394. 3. Wuchter C, et al. (2006) Archaeal nitrification in the ocean. Proc Natl Acad Sci USA 32. Schramm A, De Beer D, Wagner M, Amann R (1998) Identification and activities in situ fl 103:12317–12322. of Nitrosospira and Nitrospira spp. as dominant populations in a nitrifying uidized – 4. Reigstad LJ, et al. (2008) Nitrification in terrestrial hot springs of Iceland and bed reactor. Appl Environ Microbiol 64:3480 3485. Kamchatka. FEMS Microbiol Ecol 64:167–174. 33. Utåker JB, Andersen K, Aakra Å, Moen B, Nes IF (2002) Phylogeny and functional 5. Leininger S, et al. (2006) Archaea predominate among ammonia-oxidizing expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic – prokaryotes in soils. Nature 442:806–809. ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI. J Bacteriol 184:468 478. 6. Di HJ, et al. (2009) Nitrification driven by bacteria and not archaea in nitrogen-rich 34. Nelson KA, Moin NS, Bernhard AE (2009) Archaeal diversity and the prevalence of Crenarchaeota in salt marsh sediments. Appl Environ Microbiol 75:4211–4215. grassland soils. Nat Geosci 2:621–624. 35. Walker CB, et al. (2010) Nitrosopumilus maritimus genome reveals unique 7. Brochier-Armanet C, Boussau B, Gribaldo S, Forterre P (2008) Mesophilic mechanisms for nitrification and autotrophy in globally distributed marine Crenarchaeota: Proposal for a third archaeal phylum, the Thaumarchaeota. Nat Rev crenarchaea. Proc Natl Acad Sci USA 107:8818–8823. Microbiol 6:245–252. 36. Berg IA, et al. (2010) Autotrophic carbon fixation in archaea. Nat Rev Microbiol 8: 8. Spang A, et al. (2010) Distinct gene set in two different lineages of ammonia- 447–460. oxidizing archaea supports the phylum Thaumarchaeota. Trends Microbiol, 18: 37. Jahn U, Huber H, Eisenreich W, Hügler M, Fuchs G (2007) Insights into the autotrophic 331–340 10.1016/j.tim.2010.06.003. CO2 fixation pathway of the archaeon Ignicoccus hospitalis: Comprehensive analysis 9. Könneke M, et al. (2005) Isolation of an autotrophic ammonia-oxidizing marine of the central carbon metabolism. J Bacteriol 189:4108–4119. – archaeon. Nature 437:543 546. 38. Huber H, et al. (2008) A dicarboxylate/4-hydroxybutyrate autotrophic carbon 10. de la Torre JR, Walker CB, Ingalls AE, Könneke M, Stahl DA (2008) Cultivation of assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis. Proc Natl a thermophilic ammonia-oxidizing archaeon synthesizing crenarchaeol. Environ Acad Sci USA 105:7851–7856. – Microbiol 10:810 818. 39. Ramos-Vera WH, Berg IA, Fuchs G (2009) Autotrophic carbon dioxide assimilation in 11. Hatzenpichler R, et al. (2008) A moderately thermophilic ammonia-oxidizing Thermoproteales revisited. J Bacteriol 191:4286–4297. crenarchaeote from a hot spring. Proc Natl Acad Sci USA 105:2134–2139. 40. Berg IA, Ramos-Vera WH, Petri A, Huber H, Fuchs G (2010) Study of the distribution of 12. Wuchter C, Schouten S, Boschker HTS, Sinninghe Damsté JS (2003) Bicarbonate autotrophic CO2 fixation cycles in Crenarchaeota. Microbiology 156:256–269. uptake by marine Crenarchaeota. FEMS Microbiol Lett 219:203–207. 41. Ishii M, et al. (1996) Autotrophic carbon dioxide fixation in Acidianus brierleyi. Arch 13. Dumont MG, Murrell JC (2005) Stable isotope probing—linking microbial identity to Microbiol 166:368–371. function. Nat Rev Microbiol 3:499–504. 42. Menendez C, et al. (1999) Presence of acetyl coenzyme A (CoA) carboxylase and 14. Whitby CB, et al. (2001) (13)C incorporation into DNA as a means of identifying the propionyl-CoA carboxylase in autotrophic Crenarchaeota and indication for active components of ammonia-oxidizer populations. Lett Appl Microbiol 32:398–401. operation of a 3-hydroxypropionate cycle in autotrophic carbon fixation. J Bacteriol 15. Freitag TE, Chang L, Prosser JI (2006) Changes in the community structure and activity 181:1088–1098. of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater- 43. Berg IA, Kockelkorn D, Buckel W, Fuchs G (2007) A 3-hydroxypropionate/4- marine gradient. Environ Microbiol 8:684–696. hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. 16. Jia Z, Conrad R (2009) Bacteria rather than Archaea dominate microbial ammonia Science 318:1782–1786. oxidation in an agricultural soil. Environ Microbiol 11:1658–1671. 44. Hallam SJ, et al. (2006) Pathways of carbon assimilation and ammonia oxidation 17. Schauss K, et al. (2009) Dynamics and functional relevance of ammonia-oxidizing suggested by environmental genomic analyses of marine Crenarchaeota. PLoS Biol 4: archaea in two agricultural soils. Environ Microbiol 11:446–456. e95. 18. Offre P, Prosser JI, Nicol GW (2009) Growth of ammonia-oxidizing archaea in soil 45. Venter JC, et al. (2004) Environmental genome shotgun sequencing of the Sargasso – microcosms is inhibited by acetylene. FEMS Microbiol Ecol 70:99–108. Sea. Science 304:66 74. fi 19. Zhang L-M, et al. (2010) Autotrophic ammonia oxidation by soil thaumarchaea. Proc 46. DeLong EF, et al. (2006) Community genomics among strati ed microbial ’ – Natl Acad Sci USA 107:17240–17245. assemblages in the ocean s interior. Science 311:496 503. 20. Whiteley AS, Thomson B, Lueders T, Manefield M (2007) RNA stable-isotope probing. 47. Mincer TJ, et al. (2007) Quantitative distribution of presumptive archaeal and fi fi Nat Protoc 2:838–844. bacterial nitri ers in Monterey Bay and the North Paci c Subtropical Gyre. Environ – 21. Buyanovsky GA, Wagner GH (1983) Annual cycles of carbon dioxide level in soil air. Microbiol 9:1162 1175. 48. Bürgmann H, Widmer F, Sigler WV, Zeyer J (2003) mRNA extraction and reverse Soil Sci Soc Am J 47:1139–1145. transcription-PCR protocol for detection of nifH gene expression by Azotobacter 22. Stark JM, Hart SC (1997) High rates of nitrification and nitrate turnover in vinelandii in soil. Appl Environ Microbiol 69:1928–1935. undisturbed coniferous forests. Nature 385:61–64. 49. Neufeld JD, et al. (2007) DNA stable-isotope probing. Nat Protoc 2:860–866. 23. Neufeld JD, Dumont MG, Vohra J, Murrell JC (2007) Methodological considerations 50. Kolb S, Knief C, Dunfield PF, Conrad R (2005) Abundance and activity of uncultured for the use of stable isotope probing in microbial ecology. Microb Ecol 53:435–442. methanotrophic bacteria involved in the consumption of atmospheric methane in 24. Lueders T (2009) Stable isotope probing of hydrocarbon-degraders. Handbook of two forest soils. Environ Microbiol 7:1150–1161. Hydrocarbon and Lipid Microbiology, ed Timmis KN (Springer, Berlin), pp 4011–4026. 51. Horz HP, Barbrook A, Field CB, Bohannan BJM (2004) Ammonia-oxidizing bacteria 25. Lueders T, Manefield M, Friedrich MW (2004) Enhanced sensitivity of DNA- and rRNA- respond to multifactorial global change. Proc Natl Acad Sci USA 101:15136–15141. based stable isotope probing by fractionation and quantitative analysis of isopycnic 52. Grosskopf R, Janssen PH, Liesack W (1998) Diversity and structure of the – centrifugation gradients. Environ Microbiol 6:73 78. methanogenic community in anoxic rice paddy soil microcosms as examined by 26. Carter C, Britton VG, Haff L (1983) CsTFA: A centrifugation medium for nucleic acid cultivation and direct 16S rRNA gene sequence retrieval. Appl Environ Microbiol 64: – isolation. Biotechniques 1:142 147. 960–969. fi 27. Yakimov MM, La Cono V, Denaro R (2009) A rst insight into the occurrence and 53. Ludwig W, et al. (2004) ARB: A software environment for sequence data. Nucleic expression of functional amoA and accA genes of autotrophic and ammonia- Acids Res 32:1363–1371. oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea. Deep Sea Res Part II Top 54. Treusch AH, et al. (2005) Novel genes for nitrite reductase and Amo-related proteins Stud Oceanogr 56:748–754. indicate a role of uncultivated mesophilic Crenarchaeota in nitrogen cycling. Environ 28. Pernthaler A, Amann R (2004) Simultaneous fluorescence in situ hybridization of Microbiol 7:1985–1995. mRNA and rRNA in environmental bacteria. Appl Environ Microbiol 70:5426–5433. 55. Avrahami S, Liesack W, Conrad R (2003) Effects of temperature and fertilizer on 29. Valentine DL (2007) Adaptations to energy stress dictate the ecology and evolution of activity and community structure of soil ammonia oxidizers. Environ Microbiol 5: the Archaea. Nat Rev Microbiol 5:316–323. 691–705. MICROBIOLOGY

Pratscher et al. PNAS | March 8, 2011 | vol. 108 | no. 10 | 4175 Downloaded by guest on September 24, 2021