Community composition of ammonia-oxidizing bacteria and archaea … · 2013-03-28 · 4483 Environmental Microbiology (2008) 10(11), 2956-2965 doi:10.1111 / j.1462-2920.2008.01600.x

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Environmental Microbiology (2008) 10(11), 2956-2965 doi:10.1111 / j.1462-2920.2008.01600.x

Community composition of ammonia-oxidizing bacteriaand archaea in soils under stands of red alder andDouglas fir in Oregon

Stephanie A. Boyle-Yarwood,1* Peter J. Bottomleyt1,2

and David D. Myrold1

Departments of 1Crop and Soil Science and2Microbiology, Oregon State University, Corvallis, OR,USA.

Summary

This study determined nitrification activity and nltrl-fier community composition in soils under stands ofred alder (Alnus rubra) and Douglas fir (Pseudotsugamenziesil) at two sites in Oregon. The H.J. AndrewsExperimental Forest, located in the Cascade Moun-tains of Oregon, has low net N mineralization andgross nitrification rates. Cascade Head ExperimentalForest, in the Coast Range, has higher net Nmineralization and nitrification rates and soil pH islower. Communities of putative bacterial [ammonia-oxidizing bacteria (AOB)] and archaeal [ammonia-oxidizing archaea (AOA)] ammonia oxidizers wereexamined by targeting the gene amoA, which codesfor subunit A of ammonia monooxygenase. Nitrifica-tion potential was significantly higher in red aldercompared with Douglas-fir soil and greater atCascade Head than H.J. Andrews. Ammonia-oxidizingbacteria amoA genes were amplified from all soils,but AOA amoA genes could only be amplified atCascade Head. Gene copy numbers of AOB andAOA amoA were similar at Cascade Head regardlessof tree type (2.3-6.0 x 106amoA gene copies G-1 ofsoil). DNA sequences of amoA revealed that AOBwere members of Nitrosospira clusters 1, 2 and 4.Ammonia-oxidizing bacteria community composition,determined by terminal restriction fragment lengthpolymorphism (T-RFLP) profiles, varied among sitesand between tree types. Many of the AOA amoAsequences clustered with environmental clonespreviously obtained from soil; however, severalsequences were more similar to clones previouslyrecovered from marine and estuarine sediments. As

Received 23 April, 2007; accepted 29 January, 2008. 'Forcorrespondence. E-mail [email protected]; Tel.(+1) 5417371840; Fax (+1) 5417370496.

with AOB, the AOA community composition differedbetween red alder and Douglas-fir soils.

Introduction

Nitrogen availability and turnover vary considerablyamong different forest types within the Pacific Northwest(Hart et al., 1997; Leckie et al., 2004; Grayston and Pres-cott, 2005), and high rates of nitrification have beenobserved in forested sites where net primary productivityis high and in soils associated with red alder (Hart et al.,1997). Although the existence of acetylene-sensitive nitri-fication and the presence of ammonia-oxidizing bacteria(ADB) in soil at pH < 5 has led to speculation that ADBmay be the primary nitrifiers in acidic soils (De Boer andKowalchuk, 2001; Gieseke et al., 2006), questions remainabout the ability of ADB to nitrify under these conditionsbecause no cultured isolates can nitrify at pH < 5.

During the last few years, crenarchaeota possessingputative amoA genes have been identified in marine andterrestrial environments (Konneke et al., 2005; Treuschet al., 2005; Leininger et al., 2006). Detection and quan-tification of amoA RNA transcripts (Leininger et al., 2006)and an increase in transcript number following NH4+ addi-tions (Treusch et al., 2005) suggest that ammonia-oxidizing archaea (AOA) may function in at least someagricultural and grassland soils (Nicol and Schleper,2006). Investigations in other soil types are needed todetermine if AOA are ubiquitously distributed in soils andif their composition and activities are influenced by soilfactors and plant community composition.

In the case of AOB, Nitrosospira clusters 2, 3 and 4have been identified in acidic forest soils (Laverman et al.,2001; Mintie et al., 2003; Compton et al., 2004; Nugrohoet al., 2005; 2006) and research has linked nitrifier com-munity composition to factors such as vegetation type(Nugroho et al., 2005), microclimate (Mintie et al., 2003),disturbance (Yeager et el., 2005) and temperature (Avra-hami et al., 2003; Avrahami and Conrad, 2005). The linksbetween AOB diversity and function remain unclear,however (Kowalchuk et al., 2000; Laverman et al., 2001;Webster et al., 2002; 2005; Horz et al., 2004; Chu et al.,2007). Bottomley and colleagues (2004) found that evenwhen soil was reciprocally transferred from forest to

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Ammonia-oxidizing bacteria and archaea in forest soils 2957

at Cascade Head. A residual rate of acetylene-insensitivenitrification remained in red alder soil from Cascade Headthat was approximately 12% of the control rate (Table 1).At the end of the incubation, pH values of the soil slurriesremained similar to those measured in the soils initially(4.6-5.3 at Cascade Head and 5.2-5.8 at H.J. Andrewssoils).

Quantitative PCR determination of amoA genecopy numbers

Ammonia-oxidizing bacteria amoA copy numbers weresignificantly higher in red alder soils from H.J. Andrewsthan red alder soils from Cascade Head; at H.J. Andrewscopy numbers were significantly higher in soil under redalder compared with Douglas fir (Fig. 1). Ammonia-oxidizing archaea amoA copy numbers were below detec-tion limits (5 x 104 copies g-1 of soil) in H.J. Andrews soils.At Cascade Head AOA amoA copies were higher inDouglas-fir soil compared with red alder soil (P = 0.07).The ratios of AOA:AOB copy numbers in Cascade HeadDouglas-fir and red alder soils were 1.80 and 0.42 respec-tively (Fig. 1). Ammonia-oxidizing bacteria amoA copynumbers were higher in red alder soils compared withAOA copy numbers (P = 0.10). Copy numbers did notcorrelate with nitrification potential for either gene.

Terminal restriction fragment length polymorphism(T-RFLP) profiles and terminal restriction

fragment abundance

Restriction patterns of PCR-amplified AOB amoAsequences generated with Taql indicated that all AOBwere members of the genus Nitrosospira; all patterns

Fig. 1. amoA gene copy numbers for AOB and AOA. Barsrepresent the mean of three field replicates with SE (n = 3). Theratio of AOA:AOB is given above the bars for Cascade Head.Ammonia-oxidizing archaea copy numbers at H.J. Andrews werebelow a detection limit of 5 x 10' copies g-1 of soil.

© 2008 The AuthorsJournal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 10, 2956-2965

Mean ± standard error (n = 3). Statistical significance for main effectsof site and tree species is shown as a P-value (NS = P> 0.10);site-by-tree interactions were not significant (P< 0.05).

meadow environments and nitrification and AOB popula-tions increased, amoA composition did not significantlyvary after 2 years. In contrast, Horz and colleagues(2004) reported increased nitrification and a communityshift towards Nitrosospira cluster 2 following fertilizationexperiments. The question remains to what extent treetype or ecosystem net primary productivity may influencethe population sizes and composition of AOB in forestsoils.

A study was designed to compare the AOB and AOAcommunity compositions in soils under different treespecies and that express different rates of nitrification.Two experimental tree plantations containing pure standsof Douglas fir (Pseudotsuga menziesiJ) and red alder(Alnus rubra) were selected for these experiments. Previ-ous research had indicated differences in gross and netnitrification rates between vegetation types at each site(Boyle et al., 2008). The soil at Cascade Head Experi-mental Forest had gross nitrification rates that were three-fold higher than in soils at the H.J. Andrews ExperimentalForest. The objectives of the current study were to: (i)measure nitrification potential in soils under differing veg-etation types, and (ii) quantify and characterize the AOBand AOA communities and examine correlations betweencomposition and function.

Results

Nitrification potential

Nitrification potential varied significantly between sitesand tree types (Table 1). Cascade Head soils averaged2.69 mg of N kg-1 of soil day-1 compared with H.J.Andrews soils, which averaged 0.88 mg of N kg-1 of soilday-1 Nitrification potential was 2- and 12-fold greater inred alder compared with Douglas-fir soils at CascadeHead and the H.J. Andrews respectively. When acetylenewas added to nitrification slurries, N03- did not accumu-late in H.J. Andrews soils or in soils from under Douglas fir

2958 S. A. Boyle- Yarwood, P. J. Bottomley and D. D. Myrold

0.11) (Fig. 2A). Ammonia-oxidizing bacteria amoA com-munity composition correlated with nitrification potential(r2 = 0.41) and A08 amoA copies g-1 of soil (r2 = 0.85).Indicator species analysis identified specific terminalrestriction fragments (T-RFs) that varied based on siteand tree type. There were several T-RFs that differedsignificantly between sites, including Alul 490. Alul 490was significantly higher in Cascade Head soils than H.J.Andrews soils (Fig. 3A). In the case of tree type Cfol 95was different (P= 0.06) with a higher percentage of totalfluorescence in red alder compared with Douglas-fir soils(Fig.3A).

Archaeal amoA was amplified from all Cascade Headplots, but was not amplified from any H.J. Andrews plot.Terminal restriction fragment length polymorphism profilesfor AOA amoA were significantly different between the twotree types (P= 0.022, A-statistic = 0.43) and correlatedwith nitrification potential (r2 = 0.51) and AOA amoAcopies g-1 of soil (r2 = 0.30). Indicator species analysis didnot identify specific T-RFs that contributed to tree typedifferences at a P-value < 0.05, but several T-RFs weresignificant at P = 0.10, including Mspl 442 and Rsal 292.The per cent fluorescence of Mspl 442 was greater in redalder soil, whereas Rsal 292 was greater in Douglas-firsoil (Fig. 38).

ADB and ADA sequences

Ammonia-oxidizing bacteria clones were screened forthose T-RFs that contributed to site and tree typedifferences. Phylogenetic analysis revealed that mostsequences belonged to Nitrosospira clusters 2 and 4(Avrahami et al., 2002), with two clones A-36 (Alul 490-Cfol 136) and 8-22 (Alul 393-Cfol 167) belonging tocluster 1 (Fig. 4). Of the remaining clones, 8-18 (Alul389-Cfol 167) and 8-44 (Alul 167-Cfol 167) were closelyrelated to Nitrosospira CT2F (Mintie et al., 2003), a cluster4 isolate from a higher elevation conifer soil in the H.J.Andrews. Clones C-3 (Alul 490-Cfol 95), C-18 (Alul 490-

Fig. 2. Non-metric multidimensional scaling (NMS) ordination ofT-RFLP profiles for AOB amoA (A) and AOA amoA communities(B). Cascade Head (triangles), H.J. Andrews (circles), Douglas fir(black symbols) and red alder (grey symbols). There was noamplification of AOA amoA in H.J. Andrews soils. Vectors showcorrelation with nitrification potentials and ammonia oxidizerpopulations.

contained a single peak at 283 bp (Horz et al., 2000).Community composition determined by AOB amoA termi-nal restriction fragment length polymorphism (T-RFLP)profiles using Alul and Cfol varied by site (P= 0.02, A-statistic = 0.12) and tree type (P = 0.03, A-statistic =

Fig. 3. Relative abundance of selected T-RFsfor AOB amoA profiles (A) and AOA amoAprofiles (B). Bars represent the mean of threereplicates with SE.



Fig. 4. Bacterial amoA tree constructed usingBayesian anaiysis. Numbers show theprobability of clade assignments. Clusterassignments are made based on Avrahamiand colleagues (2002). Environmental clonesfrom this study appear in bold with the site,Cascade Head (CH) or H.J. Andrews (HJA).and tree type, Douglas fir (OF) or red alder(RA). appearing in parentheses.

Cfol 95) and C-23 (Alul 393-Cfol 95) all contained cut siteCfol 95, the dominant T-RF in red alder soils (Figs 3Aand 4).

Twenty archaeal amoA sequences were used in a phy-logenetic analysis. At 98% similarity the clone library con-tained 15 unique operational taxonomic units (OTUs).Clones clustered into four main clades. Clones 12C-1,48-2, 4C-2, 5C-1 and 9H-1 (Cfol 261-Mspl 559-Rsal 292)

were 99% similar and grouped closely with other environ-mental clones from soil (Fig. 5). Clones 12C-2, 4A-1 and7F-1 (Cfol 559-Mspl 258-Rsal 559) were 98% similar andalso grouped with soil clones. The remaining AOAsequences including clone 12F-2 (Cfol 559-Mspl 442-Rsal 203) were more divergent and tended to cluster withclones recovered from estuarine sediments, marine andwastewater treatment plants (Francis et al., 2005) (Fig. 5).



Fig. 5. Archaeal amoA tree constructed usingBayesian analysis. Numbers show theprobability of clade assiqnments.Environmental clones from this study appearin bold with the site Cascade Head (CH) andtree type, Douglas fir (OF) or red alder (RA),appearing in parentheses.

Discussion

The presence of red alder in conifer-dominated ecosys-tems has been shown to increase both gross and netnitrification in soil (Binkley et al., 1992; Hart et al., 1997;Boyle et al., 2008), and reduce soil pH (Rhoades andBinkley, 1992). Data from both experimental sites showedgreater nitrification potential in red alder compared withDouglas-fir soils, although the nitrification potentialincreased to a greater extent under red alder at H.J.Andrews, where red alder may have contributed to aproportionately greater increase in available N. The nitri-fication potential rates measured in this study, using soil


slurries, were similar to the rates of gross nitrificationmeasured in whole soil by Boyle and colleagues (2008)(r2 = 0.50) (Table 1), suggesting that at the time of sam-pling nitrification conditions may have been close tooptimum even at the low pH of whole soil and nitrificationpotential soil slurries.

Based on pure culture studies of AOB that determinedrates of NH3 oxidation to be between 1 and 10 x 10-15 molcell-1 h-1 (Belser and Schmidt, 1978; Jiang and Bakken,1999), the nitrification potentials measured in these soilscould be supported by AOB population densities as low as0.4-4.0 x 105 cells g-1 of soil in H.J. Andrews Douglas-firsoil and as high as 0.1-1.1 x 10-7 cells g-1 of soil in


Cascade Head red alder soil. If we assume three copiesof amoA cell-1 (J. Norton, unpublished), these populationvalues fall within estimates made by quantitative PCR(Q-PCR) (Fig. 1) and also agree with values reportedelsewhere (Okano et al., 2004; Leininger et al., 2006).In Douglas-fir soils, differences in gene copy numbersbetween sites correlated with nitrification potential(Table 1, Fig. 1), but no correlation was found for red aldersoil. Furthermore, in Cascade Head red alder soils, thepopulation densities (2.0 x 106 cells g-1 of soil) were at thelower limit of the range that we predicted based on nitri-fication potentials, suggesting that if AOB are solelyresponsible for nitrification in these soils they are oxidizingNH3 close to the highest rate documented for pure cul-tures (10 x 10-15 mol cell-1 h-1). Although it is possible toaccount for all nitrification activity with measured AOBpopulations these findings provide circumstantial evi-dence that another population of organisms may be con-tributing to NH3 oxidation.

The AOA: AOB ratios measured at Cascade Head(Fig. 2) tended to be lower (1.8 and 0.42) than thosereported previously in European soils (1.5-232) (Lein-inger et al., 2006), perhaps because the soils at CascadeHead are forested, more acidic (3.6-4.1), and containhigher concentrations of organic nitrogen (Boyle et al.,2008). To the authors' knowledge there are no publishedreports of rates of NH3 oxidation per cell by cultures ofAOA. Using data presented by K6nneke and colleagues(2005) we calculated that the rate of NH3 oxidation byNitrosopumilus maritimus ranges between 0.25 and

0.35 x 10-15 mol cell -1 h-1, if we assume that soil AOApossess one copy of amoA cell-1 like N. meritimus (Stahland Richardson, 2007). The AOA population density esti-mates made at Cascade Head of approximately 107 cellsg-1 of soil could support the nitrification potentials mea-sured at the site. Further research is needed to determinethe relative contribution of AOA and AOB to nitrification inthese soils.

Ammonia-oxidizing bacteria composition determined byT-RFLP profiles varied by site and tree type, suggestingthat complex changes brought about by differing climate,soil and vegetation types affected the diversity of AOB.Terminal restriction fragment length polymorphism profilesalso correlated with AOB copy number, but correlated toa lesser extent with nitrification potential. These resultscould be due to T-RFLP profiles being based on total AOBcommunity DNA, which encompasses both active andinactive members of the oxidizer community. Terminalrestriction fragment Cfol 95 was significantly higher in redalder compared with Douglas-fir soils (Fig. 3), and at leastone clone (C-3) that contained Cfol 95 appeared tobelong to Nitrosospira cluster 2. Previously, researchersidentified Nitrosospira cluster 2 in chronic N-amendmentplots (Compton et al., 2004) and acidic Scots pine forests

(Nugroho et al., 2005). Although Nugroho and colleagues(2007) suggested that cluster 2 may survive in low pHsoils through urea-hydrolysis, urease is not unique toNitrosospira cluster 2 and is widely, albeit non-uniformlydistributed among AOB (Koops and Pommerening-R6ser,2001; Koops et al., 2003). Further work is needed tounderstand why Nitrosospire cluster 2 thrives in thisacidic, high N environment. It is also worth mentioningthat two of the clones recovered from the H.J. Andrewsalso grouped with Nitrosospira cluster 1, a group foundpredominately in marine systems. Because no isolatesexist for Nitrosospira cluster 1, their role in NH3 oxidationremains speculative.

We believe our data are the first to show the presenceof AOA amoA in forest soils under both coniferous anddeciduous tree species. Archaeal amoA did not amplifyeven after several attempts in H.J. Andrews soils withdifferent primer sets (Francis et al., 2005; Treusch et al.,2005) and under different conditions, suggesting that AOAmay exist at levels below our current amplification ability.Alternatively, if present, AOA in H.J. Andrews soils may besufficiently different so as not to amplify with currentprimers. Given that we were able to recover sequencesfrom Cascade Head that grouped with a variety of otherknown sequences, H.J. Andrew AOA could be signifi-cantly different from those in the current public database.Ammonia-oxidizing archaea amoA consistently amplifiedin all Cascade Head soils, and T-RFLP profiles from puta-tive AOA amoA genes at Cascade Head differed by treetype (Fig. 2B), suggesting that AOA community composi-tion, like AOB composition, may be impacted by changesbrought about by the presence of red alder. Terminalrestriction fragment Rsal 292 was higher in Douglas-firsoil (Fig. 3B) and was found in a group of clones thatclustered with other AOA amoA soil clones, whereas Mspl442 was greater in red alder soil and was observedin sequences that clustered with sediments. As moresequences of AOA amoA are collected and made avail-able, it will be interesting to see if AOA sequences con-tinue to cluster based on environment or if other factorssuch as vegetation type or N availability will shed newlight on the factors influencing their community com-position and activity. Our unpublished findings also indi-cated that archaeal communities based on 16S rRNAsequences differed between Cascade Head and H.J.Andrews soils and between tree types. Other researchhas also shown that archaeal populations may varybetween soil types and plant communities (Jurgenset al., 1997; Ochsenreiter et al., 2003; Nicol et al., 2007).Further research is needed to determine if differences inAOA distribution, population size and community compo-sition contribute to the increased rates of NH3 oxidationthat we observed in soils at Cascade Head and in thepresence of red alder.


2962 S. A Boyle- Yarwood, P. J. Bottomley and D. D. Myrold

Experimental procedures

Site description and sample collection

Soils were sampled in two experimental tree plantationswhere trees were approximately 20 years old (Radosevichet al., 2006). Plots measured 27 m x 27 m and containedeither pure stands of Douglas fir or red alder. The first sitewas located in Cascade Head Experimental Forest, 1.6 kmfrom the Pacific Ocean, at an elevation of 330 m. The weath-ering of basaltic headlands has created a Histic Epiaquand(Rhoades and Binkley, 1992) with high soil fertility and pH 4.The second site was located in the H.J. Andrews Experimen-tal Forest in the Cascade Mountains at an elevation of 800 m.The soil is best classified as a Haplumbrept (Dyrness, 2005)with pH 5. Concentrations of soil N, NH: and NO3

- are sig-nificantly lower at H.J. Andrews in comparison with CascadeHead (Boyle et al., 2008). Soil NO3

- concentrations averaged6.2 mg kg-1 of soil at Cascade Head and 1.8 mg kg-1 of soil atH.J. Andrews. Red alder soils had significantly higher NO3

-

concentrations (Boyle et al., 2008), with the highest NO3-

concentrations in Cascade Head red alder soil (8.0 mg kg-1of soil) and the lowest in H.J. Andrews Douglas-fir soil(0.3 mg kg-1 of soil). Soil samples were collected in the springof 2006, 1 month after red alder leaves emerged. Ten soilcares (3 c m x 10 cm) were taken from each of three replicateplots per treatment. Five cores were placed into each of twobags per plot and homogenized. Samples were transportedon ice to the lab, where 1 g of samples were removed fromeach bag for DNA extraction. The remaining soil from eachplot was homogenized and used in nitrification potentialassays.

Nitrification potential assay

Nitrification potentials were determined on fresh soils using ashaken soil-slurry method (Hart et al., 1994). After sieving(4 mm), portions of soil (15 g of dry-weight equivalent) fromeach plot were extracted with 135 ml of 1.0 mM potassiumphosphate buffer (pH 7.2) in order to remove backgroundNO3-. The slurries were then centrifuged (6000 g; 10 min) topellet soil particles and the buffer was decanted. The soilpellet was re-suspended in 135 ml of 1.0 mM phosphatebuffer (pH 7.2) containing 1.5 mM NH:. Aliquots of the soilsuspension (20 ml) were placed into six, 160 ml serum vials,taking care to keep the slurry well mixed. The remainingslurry was centrifuged to pellet soil particles and filtered(211m). This filtrate was used to establish initial NO3-

concentrations. The six serum vials were crimp-sealed usingbutyl rubber stoppers and acetylene was added to three ofthe vials (10.8 kPa). All vials were shaken at 25°C for 30 h,centrifuged to remove soil particles, and filtered (211m). Con-centrations of NO3- in the filtrates were measured using anautoanalyser (Astoria Pacific, Portland, OR). The pH of thesoil slurries was measured at the end of the incubation.

DNA extractions and PCR amplification

DNA was extracted from soil (0.25 g) using an MOBio Pow-erSoWMDNA isolation kit (MoBio Laboratories, Carlsbad, CAlaccording to the manufacturer's instructions, with the modifi-

cation that a Bio101 FastPrepTM instrument was used to lysecells (MP Biomedicals, Solon, OH). The MOBio bead beatingtubes were shaken for 45 s using the FastPrep instrument.Extracts were quantified using a NanoDropTM ND-1000UV-Vis Spectrophotometer (NanoDrop Technologies, Wilm-

ington, DE) and diluted to 25 ng µl-1. Two extracts from eachplot were used as a composite template by combining 25 µl ofeach 25 ng µl-1 dilution.

Approximately 100 ng of DNA was used in each conven-tional PCR reaction. Ammonia-oxidizing bacteria amoAgenes were amplified using primers amoA-1 F (5'-GGG GGTTIC TAC TGG TGG T) and amoA-2R (5'-CCC CTC KGSAAA GCC TIC TIC) (Harz et al., 2000), under conditionsdescribed previously (Mintie et al., 2003) using a PTC-100Programmable Thermocycler (MJ Research, Watertown,MA). Archaeal amoA was amplified using primers amo111F(5'-TTY TAY ACH GAY TGG GCH TGG ACA TC) andam0643R (5'-TCC CAC TTW GAC CAR GCG GCC ATC CA)(Treusch et al., 2005) in a 50 µl reaction mix containing: 1xPCR buffer, 2 mM MgCI2 0.2 mM dNTPs, 0.2 µ Primer,0.064% BSA. Archaeal amoA was amplified using the follow-ing conditions: 94°C far 5 min, followed by 30 cycles at 94°Cfor 30 s, 55°C far 30 s, 72°C for 45 s, ending with an exten-sion step of 72°C for 7 min. Additional PCR conditions weretested with DNA extracts from H.J. Andrews in an attempt toachieve amplification. A DNA EngineTM thermocycler (Bio-Rad Laboratories, Hercules, CA) with a gradient block wasused to test annealing temperatures from 45°C to 55°C andcycle numbers of 30, 35 and 40. Although these less stringentconditions resulted in non-specific amplification in CascadeHead soils, no amplification of the correct size fragment wasobserved in H.J. Andrews soils.

T-RFLP and Q-PCR

Terminal restriction fragment length polymorphism profileswere generated using primers amoA-1 F (Harz et al., 2000;Mintie et al., 2003) and amo111F (Treusch et al., 2005)labelled with a 6-FAM fluorophors. The resulting productswere cleaned using a QiaquickTM PCR Purification kit(Qiagen, Valencia, CA) and restricted. Ammonia-oxidizingbacteria PCR products were restricted using Alul, Cfol andTaql, and AOA PCR products were restricted with Cfol, Mspland Rsal. Restriction products were column-purified and pro-files were analysed by the Oregon State University Centerof Genome Research and Biocomputing using an ABI3100 capillary DNA sequencer (Applied Biosystems, FosterCity, CA).

Ammonia-oxidizing bacteria and AOA amoA copy numberswere determined using methods described previously (Lein-inger et al., 2006). Briefly, primers amoA-1 F and amoA-2R incombination with the Brilliant SYBR GreenTM Q-PCR CoreReagent Kit (Stratagene, La Jolla, CAl were used in AOBquantification and primers amo196F and amo277R with theprobe amo247 labelled with 6-FAM (Treusch et al., 2005)were used in AOA quantification. All assays were run on anABI 7500 Sequence detection system (Applied Biosystems,Foster City, CA). Each reaction contained 2 µl of a1.25 ng µl-1 dilution and was run in triplicate. Genomic DNAfrom Nitrosomonas europaea at a range of 5.0 x 10-1 to5.0 x 10-7 ng of DNA per reaction was used to generate a

© 2008 The AuthorsJournal compilation© 2008 Society for Applied Microbiologyand Blackwell Publishing Ltd, Environmental Microbiology, 10, 2956-2965


standard curve for AOB assays. Plasmid DNA from an AOAenvironmental clone at a range of 5.0 x 10-1to 5.0 x 10-5 ngof DNA per reaction was used to generate AOA standardcurves. Standard curves from each run were analysed toverify r2 values> 0.95 and efficiency values between 95%and 105%. Detection limits for AOB and AOA amoA Q-PCRassays were 3 x 103 copies g-1of soil and 5 x 104 copies g-1

of soil respectively.

Cloning and sequencing

Clones were generated using a Topo TA cloningTM kit forsequencing (Invitrogen, Carlsbad, CA) according to manufac-turer's instructions. Ammonia-oxidizing bacteria clones werescreened by PCR amplification and T-RFLP profiles. A total of30 AOA clones and 24 AOB clones were sequenced by theHigh Throughput Genomics Unit (Department of GenomeScience, University of Washington, Seattle, WA).

Statistical and phylogenetic analysis

Univariate statistical analyses were performed using SAS 9.1(SAS Institute, Cary, NC). Nitrification potential was analysedby two-way analysis of variance (ANOVA) (Ramsey andSchafer, 2002). Where interactions occurred, data wereanalysed separately and contrasts were used to test specifictreatment differences. Linear regression analysis was alsoperformed to determine if population size or compositioncorrelated to nitrification activity. P-values < 0.05 were con-sidered significant.

Quantitative PCR data were analysed using ABI Sequencedetection system software version 1.4 (Applied Biosystems,Foster City, CA) and T-RFLP profiles were analysed usingGenoTyper version 3.7 (Applied Biosystems, Foster City,CA). Terminal restriction fragment length polymorphism pro-files were further analysed according to methods describedpreviously (Boyle et al., 2006) and compositional differenceswere investigated using PC-ORO Multivariate Analysis ofEcological Data version 5.0 (MjM software, Gleneden Beach,OR). Non-metric multidimensional scaling (NMS) was used toevaluate community compositional variation and multire-sponse permutation procedures (MRPP) were used to deter-mine differences between site and tree type (McCune andGrace, 2002).

DNA sequences were aligned using CLUSTALX version 1.81(Thompson et al., 1997) and alignments were edited usingBioedit sequence alignment editor version 7.0.5 (Hall, 1999).All sequences were analysed using Mallard Version 1.02(Ashelford et al., 2006) to ensure that no chimeras or othersequencing anomalies occurred. Phylogenetic trees wereconstructed using Mr Bayes Version 3.1 (Huelsenbeck andRonquist, 2001; Ronquist and Huelsenbeck, 2003) andconfirmed using Phylip Version 3.2 (Felsenstein, 1989). MrBayes was run using an omega variation model (M3), acodon model that calculates the likely rate of variation at eachsite. The model was run for 1 million generations to ensureconvergence at a stable value (Hall, 2001). Terminal restric-tion cut sites were determined for each sequence usingBioedit alignment editor and confirmed using Webcutter 2.0(Heiman, 1997).

Acknowledgements

Support was provided by grants from the USDA NRICGP, theNational Science Foundation IGERT Program and the H.J.Andrews LTER fund. Thanks go to Dr Rockie Yarwood andElizabeth Brewer for assistance in sample collection andpreparation, to Oregon State University's Center for GenomeResearch and Biocomputing for use of Q-PCR facilities andgenotyping analysis, and to the University of WashingtonHigh Throughput Genomic Sequencing Unit for sequenceanalysis.

References

Ashelford, K.E., Chuzhanova, NA, Fry, J.C., Jones, AJ.,and Weightman, AJ. (2006) New screening softwareshows that most recent large 16S rRNA gene clone librar-ies contain chimeras. Appl Environ Microbial 72: 5734-5741.

Avrahami, S., and Conrad, R. (2005) COld-temperatureclimate: a factor for selection of ammonia oxidizers inupland soil? Can J Microbiol51: 709-714.

Avrahami, S., Conrad, R., and Braker, G. (2002) Effect of soilammonium concentration on N20 release and on commu-nity structure of ammonia oxidizers and denitrifiers. ApplEnviron Microbio/68: 5685-5692.

Avrahami, S., Liesack, W., and Conrad, R. (2003) Effects oftemperature and fertilizer on activity and community struc-ture of soil ammonia oxidizers. Environ Microbiol5: 691-705.

Belser, LW., and Schmidt, E.L. (1978) Diversity in theamonia-oxidizing nitrifier population of a soil. Appl EnvironMicrobio/36: 584-588.

Binkley, D., Sollins, P., Bell, R., Sachs, D., and Myrold, D.(1992) Biogeochemistry of adjacent conifer and alder-conifer stands. Ecology 73: 2022-2033.

Bottomley, P.J., Taylor, AE., Boyle, SA, McMahon, S.K.,Rich, J.J., Cromack, K., Jr, and Myrold, D.O. (2004)Responses of nitrification and ammonia-oxidizing bacteriato reciprocal transfers of soil between adjacent coniferousforest and meadow vegetation in the Cascade Mountainsof Oregon. Microb Eco/48: 500-508.

Boyle, SA, Rich, J.J., Bottomley, P.J., Cromack, K., andMyrold, D.O. (2006) Reciprocal transfer effects on denitri-fying community composition and activity at forest andmeadow sites in the Cascade Mountains of Oregon. SoilBioI Biochem 38: 870-878.

Boyle, SA, Yarwood, R.R., Bottomley, P.J., and Myrold, D.O.(2008) Bacterial and fungal contributions to soil nitrogencycling under Douglas-fir and red alder at two sites inOregon. Soil BioI Biochem 40: 443-451.

Chu, H., FUjii, T., Morimoto, S., Lin, X., Yagi, K., Hu, J., andZhang, J. (2007) Community structure of ammonia-oxidizing bacteria under long-term application of mineralfertilizer and organic manure in sandy loam soil. ApplEnviron Microbial 73: 485-491.

Compton, J.E., Watrud, L.S., Porteous, LA, and Degrood, S.(2004) Response of soil microbial biomass and communitycomposition to chronic nitrogen additions at HarvardForest. For Ecol Manage 196: 143-158.

De Boer, W., and Kowalchuk, G.A. (2001) Nitrification in acid

© 2008 The AuthorsJournal compilation© 2008 Society for Applied Microbiologyand Blackwell PublishingLtd, EnvironmentalMicrobiology,10, 2956--2965


soils: micro-organisms and mechanisms. Soil BioI Biochem33: 853-866.

Dyrness, C. (2005) Soil Descriptions and Data for SoilProfiles in the Andrews Experimental Forest, SelectedReference Stands, Research Natural Areas, and Na-tional Parks: Long-Term Ecological Research. Corvatlls,OR: Forest Science Data Bank: SP001. [WWW document].URL http://www.fsl.orst.edu/lter/data/abstract.cfm?dbcode=SP001.

Felsenstein, J. (1989) HYLIP - Phylogenetic InferencePackage (Version 3.2). Cladistics 5: 164-166.

Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, AE., andOakley, B.B. (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of theocean. Proc Nat! Acad Sci USA 41: 14683-14688.

Gieseke, A, Tarre, S., Green, M., and de Beer, D. (2006)Nitrification in a biofilm at low pH values: role of in situmicroenvironments and acid tolerance. Appl EnvironMicrobiol72: 4283-4292.

Grayston, S.J., and Prescott, C.E. (2005) Microbialcommunities in forest floors under four tree speciesin coastal British Columbia. Soil Bioi Biochem 37: 1157-1167.

Hall, T.A. (1999) BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.

Hall, B.G. (2001) Phylogenetic Trees Made Easy. Sunder-land, MA, USA: Sinauer Associates.

Hart, S.C., Stark, J.M., Davidson, E.A, and Firestone, M.K.(1994) Nitrogen mineralization, immobilization, andnitrification. In Methods of Soil Analysis, Part 2. Microbio-logical and Biochemical Properties. Weaver, RW., Angle,S., Bottomley, P., Bezdicek, D., Smith, S., Tabatabai, A,and Wollum, A (eds). Madison, NJ, USA: Soil ScienceSociety of America, pp. 985-1018.

Hart, S.C., Binkley, D., and Perry, DA (1997) Influence ofred alder on soil nitrogen transformations in two coniferforests of contrasting productivity. Soil Bioi Biochem 29:1111-1123.

Heiman, M. (1997) Webcutter 2.0. [WWW document]. URLhttp://rna.lundberg .gu.se/cutter2/.

Horz, H.P., Rotthauwe, J.H., Lukow, T., and Liesack, W.(2000) Identification of major subgroups of ammonia-oxldlzlnq bacteria in environmental samples by T-RFLPanalysis of amoA PCR products. J Microbiol Methods 39:197-204.

Horz, H.P., Barbrook, A, Field, C.B., and Bohannan, B.J.M.(2004) Ammonia-oxidizing bacteria respond to multifacto-rial global change. Proc Natl Acad Sci USA 101: 15136-15141.

Huelsenbeck, J.P., and Ronquist, F. (2001) MRBAYES:Bayesian inference of phylogeny. Bioinformatics 17: 754-755.

Jiang, Q.Q., and Bakken, L.R. (1999) Comparison of Nitro-sospira strains isolated from terrestrial environments.FEMS Microbial Ecol 30: 171-186.

Jurgens, G., Lindstrom, K., and Sanno, A (1997) Novelgroup within the kingdom Crenarchaeota from boreal forestsoil. Appl Environ Microbiol63: 803-805.

Konneke, MA, Bernhard, E., de la Torre, J.R., Walker, C.B.,Waterbury, J.B., and Stahl, DA (2005) Isolation of an

autotrophic ammonia-oxidizing marine archaeon. Nature437: 543-546.

Koops, H.-P., and Pornmerenlnq-Hoser, A (2001) Distribu-tion and ecophysiology of the nitrifying bacteria emphasiz-ing cultured species. FEMS Microbial Ecol37: 1-9.

Koops, H.-P., Purkhold, U., Pornrnerenlnq-Hoser, A, Tim-mermann, G., and Wagner, M. (2003) The Iithotrophicammonia-oxidizing bacteria. In The Prokaryotes. Dworkin,M., Falkow, S., Rosenberg, E., Schleifer, K.-H., and Stack-ebrandt, E. (eds). New York, NY, USA: Springer-Verlag,pp. 778-811.

Kowalchuk, GA, Stienstra, AW., Heilig, G.H.J., Stephen,J.R., and Woldendorp, JW. (2000) Changes in the com-munity structure of ammonia-oxidizing bacteria duringsecondary succession of calcareous grasslands. EnvironMicrobiol2: 99-110.

Laverman, AM., Speksnijder, AG.C.L., Braster, M., Kowal-chuk, GA, Verhoef, HA, and van Verseveld, H.W. (2001)Spatiotemporal stability of an ammonia-oxidizing commu-nity in a nitrogen-saturated forest soil. Microb Ecol 42:35-45.

Leckie, S.E., Prescott, C.E., Grayston, S.J., Neufeld, J.D.,and Mohn, W.W. (2004) Characterization of humus micro-bial communities in adjacent forest types that differ in nitro-gen availability. Microb Ecol48: 29-40.

Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J.,Nicol, G.W., et al. (2006) Archaea predominate amongammonia-oxidizing prokaryotes in soils. Nature 442: 806-809.

McCune, B., and Grace, J.B. (2002) Analysis of EcologicalCommunities. Gleneden Beach, OR, USA: MjM Software.

Mintie, AT., Heichen, R.S., Cromack, K., Myrold, D.O., andBottomley, P.J. (2003) Ammonia-oxidizing bacteria alongmeadow-to-forest transects in the Oregon CascadeMountains. Appl Environ Microbiol69: 3129-3136.

Nicol, GW., and Schleper, C. (2006) Ammonia-oxidising Cre-narchaeota: important players in the nitrogen cycle?Trends Microbiol14: 207-212.

Nicol, GW., Campbell, C.D., Chapman, S.J., and Prosser,J.1. (2007) Afforestation of moorland leads to changes increnarchaeal community structure. FEMS Microbiol Ecol60: 51-59.

Nugroho, RA, Roling, W.F.M., Laverman, AM., Zoomer,H.R., and Verhoef, HA (2005) Presence of Nitrososplrecluster 2 bacteria corresponds to N transformation rates innine acid Scots pine forest soils. FEMS Microbiol Ecol 53:473-481.

Nugroho, A.A, Roling, W.F.M., Laverman, AM., andVerhoef, HA (2006) Net nitrification rate and presence ofNitrosospira cluster 2 in acid coniferous forest soils appearto be tree species specific. Soil Biol Biochem 38: 1166-1171.

Nugroho, RA, Roling, W.F.M., Laverman, AM., andVerhoef, HA (2007) Low nitrification rates in acid Scotspine forest soils are due to pH-related factors. Microb Ecol53: 89-97.

Ochsenreiter, T., Selezi, D., Ouaiser, A, Bench-Osmolovskaya, L., and Schleper, C. (2003) Diversity andabundance of Crenarchaeota in terrestrial habitats studiedby 16S RNA surveys and real time PCR. Environ Microbiol5: 787-797.

© 2008 The AuthorsJournal compilation© 2008 Society for Applied Microbiologyand BlackwellPublishingLtd, EnvironmentalMicrobiology,10, 2956-2965

http://www.fsl.orst.edu/lter/data/abstract.cfm?dbcode

http://rna.lundberg


Okano, Y., Hristova, K.R., Leutenegger, C.M., Jackson, L.E.,Denison, R.F., Gebeyesus, B., et al. (2004) Application ofreal-time PCR to study effects of ammonium on populationsize of ammonia-oxidizing bacteria in soil. Appl EnvironMicrobiol70: 1008-1016.

Radosevich, S.R., Hibbs, D.E., and Ghersa, C.M. (2006)Effects of species mixtures on growth and stand develop-ment of Douglas-fir and red alder. Can J For Res 35:768-782.

Ramsey, F.L., and Schafer, DW. (2002) The analysis ofvariance for Two-way Classification. In The StatisticalSleuth. Crockett, C., and Jenkins, J. (eds). Pacific Grove,CA, USA: Duxbury Thomson Learning, pp. 374-400.

Rhoades, C.C., and Binkley, D. (1992) Spatial extent ofimpact of red alder on soil chemistry of adjacent coniferstands. Can J For Res 22: 1434-1437.

Ronquist, F., and Huelsenbeck, J.P. (2003) Mr. Bayes 3:Bayesian Phylogenetic inference under mixed models. Bio-informatics 19: 1572-1574.

Stahl, D., and Richardson, P. (2007) DOE Joint GenomeInstitute: Nitrosopumilus maritimus SCMI. [WWWdocument]. URL http://genome.ornl.gov/microbial/nmar.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F.,and Higgins, D.G. (1997) The CLUSTALX windows interface:flexible strategies for multiple sequence alignment aided byquality analysis tools. Nucleic Acids Res 24: 4876-4882.

Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C.,Klenk, H.-P., and Schleper, C. (2005) Novel genes fornitrite reductase and Amo-related proteins indicate a role ofuncultivated mesophilic crenarchaeota in nitrogen cycling.Environ Microbial 7: 1985-1995.

Webster, G., Embley, T.M., and Prosser, J.I. (2002) Grass-land management regimens reduce small-scale heteroge-neity and species diversity of β-Proteobacterial ammoniaoxidizer populations. Appl Environ Microbiol58: 20-30.

Webster, G., Embley, T.M., Freitag, T.E., Smith, Z., andProsser, J.I. (2005) Links between ammonia oxidizerspecies composition, functional diversity and nitrificationkinetics in grassland soils. Environ Microbiol7: 676-684.

Yeager, C.M., Northup, D.E., Grow, C.C., Barns, S.M., andKuske, C.R. (2005) Changes in nitrogen-fixing andammonia-oxidizing bacterial communities in soil of a mixedconifer forest after wildfire. Appl Environ Microbiol 71:2713-2722.

© 2008 The AuthorsJournal compilation© 2008 Society for Applied Microbiologyand Blackwell PublishingLtd, EnvironmentalMicrobiology,10, 2956-2965

http://genome.ornl.gov/microbial/nmar.

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