Phylogenetic Diversity of Archaea and the Archaeal Ammonia ...

Research ArticlePhylogenetic Diversity of Archaea and the ArchaealAmmonia Monooxygenase Gene in Uranium Mining-ImpactedLocations in Bulgaria

Galina Radeva12 Anelia Kenarova3 Velina Bachvarova2 Katrin Flemming1 Ivan Popov4

Dimitar Vassilev5 and Sonja Selenska-Pobell1

1 Helmholtz-Centre Dresden-Rossendorf Institute of Resource Ecology Bautzner Landstraszlige 400 01328 Dresden Germany2 Institute of Molecular Biology Bulgarian Academy of Sciences Academic G Bonchev Street Building 21 1113 Sofia Bulgaria3 Faculty of Biology Sofia University 8 Dragan Tsankov Boulevard 1164 Sofia Bulgaria4Molecular Medicine Centre Medical University of Sofia 1 Georgy Sofiiski Street 1431 Sofia Bulgaria5 Agrobioinstitute 8 Dragan Tsankov Boulevard 1164 Sofia Bulgaria

Correspondence should be addressed to Galina Radeva gradevabio21basbg

Received 29 November 2013 Accepted 17 January 2014 Published 11 March 2014

Academic Editor William B Whitman

Copyright copy 2014 Galina Radeva et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Uraniummining andmilling activities adversely affect the microbial populations of impacted sitesThe negative effects of uraniumon soil bacteria and fungi are well studied but little is known about the effects of radionuclides and heavy metals on archaea Thecomposition and diversity of archaeal communities inhabiting the waste pile of the Sliven uraniummine and the soil of the Buhovouranium mine were investigated using 16S rRNA gene retrieval A total of 355 archaeal clones were selected and their 16S rDNAinserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types All evaluatedarchaeal 16S rRNA gene sequences belong to the 11bNitrososphaera cluster of Crenarchaeota The composition of the archaealcommunity is distinct for each site of interest and dependent on environmental characteristics including pollution levels Since themembers of 11bNitrososphaera cluster have been implicated in the nitrogen cycle the archaeal communities from these sites wereprobed for the presence of the ammoniamonooxygenase gene (amoA) Our data indicate that amoA gene sequences are distributedin a similar manner as in Crenarchaeota suggesting that archaeal nitrification processes in uranium mining-impacted locationsare under the control of the same key factors controlling archaeal diversity

1 Introduction

Metagenomic studies have revealed that Archaea are widelydistributed and likely play an important role in a variety ofenvironmental processes such as chemoautotrophic nitrifi-cation [1] carbon metabolism [2] and amino acid uptake [34] The most abundant organisms among the archaeal phylaare Crenarchaeota and Euryarchaeota [2 5] Crenarchaeotarepresent more than 75 of the archaeal populations innatural environments [6] Certain crenarchaeotic groups arethought to be confined to specific environments for examplegroup 11a consists mainly of aquatic organisms while themembers of group 11b are typical soil crenarchaeotes [7]

Worldwidemining andmilling activities have introducedhigh levels of radionuclides and heavymetals (HMs) into soil

and aquatic environments The adverse effects of pollutantson Archaea are not well studied [8 9] Moreover only a fewstudies have investigated archaeal diversity in HM- [10 11]and uranium- (U-) contaminated environments [5 12ndash14]Radeva and Selenska-Pobell [13] reported crenarchaeotic 16SrRNA gene sequences in U-contaminated soils of SaxonyGermany belonging only to the 11b group of the phyla whileReitz et al [14] identified 11a 13b and SAGMCG1 cren-archaeotic gene sequences from deeper U-polluted soil hori-zons Porat et al [5] investigated the diversity of archaealcommunities frommercury- andU-contaminated freshwaterstream sediments by pyrosequencing analysis They found ahigher abundance and diversity of Archaea in mercury- thanin U-contaminated sites where the archaeal sequences wereof both the Crenarchaeota and Euryarchaeota phyla

Hindawi Publishing CorporationArchaeaVolume 2014 Article ID 196140 10 pageshttpdxdoiorg1011552014196140

2 Archaea

To date little is known concerning the interactionsbetween archaea and U or HMs Kashefi et al [15] pub-lished that the hyperthermophilic crenarchaeote Pyrobacu-lum islandicum is able to reduce U(VI) to U(IV) under anaer-obic conditions at 100∘C Francis et al [16] demonstratedthat the halophilic euryarchaeote Halobacterium halobiumaccumulates high amounts of U(VI) as extracellular uranylphosphate deposits however these two organisms are notfound in U-contaminated substrata Later Reitz et al [9 17]revealed the capacity of the acidothermophilic Sulfolobusacidocaldarius which is an indigenous archaeon for U-contaminated soils and mine tailings to accumulated intra-cellular U(VI)

Thediscovery that somemesophilic archaea fromCrenar-chaeota which were later categorized into the newThaumar-chaeota phylum [18] have the potential to oxidize ammoniasuggests an important role of archaea in the nitrogen (N)cycle [19 20] The crenarchaeotic ammonia monooxygenasegene (amoA) is found in many natural environments suchas soil [2 21] marine and freshwater ecosystems [22ndash25]several geothermal environments and hot springs [26ndash28]Artic lakes [29] drinking water production plants [30] andwastewater treatment plants [31] This widespread distri-bution indicates the ubiquity and significance of archaealammonia oxidizers in the global N cycle [21 32ndash34] How-ever there are few studies assessing the abundance of archaealamoA and its diversity in U-impacted environments

Intensive U mining and milling in Bulgaria were per-formed between 1946 and 1990 and have caused significantsoil and water pollution U production was stopped by agovernment decree in 1992 andmines and tailings were tech-nically liquidated and gradually remediated Neverthelesstheir surroundings are still highly contaminated and furthercontamination from the compromised remediation of minesand tailings has been recorded

The aim of this study was to investigate the diversity ofarchaeal communities inhabiting environments impacted byU mining and milling activities and in particular to revealthe diversity of the archaeal amoA gene Since U and HMcontamination represent an old environmental burden weexpected that the composition and diversity of archaeal andamoA communities were stabilized under the selective powerof both contamination level and environmental characteris-tics

2 Materials and Methods

21 Sites and Sampling Two locations in Bulgaria were stud-ied the abandoned mining and milling complex ldquoBuhovordquoand the ldquoSlivenrdquo mine both of which have been classified asareas of high radiological risk by the Bulgarian Agency forRadiobiology and Radioprotection The mining complexldquoBuhovordquo (42∘451015840512010158401015840N 23∘341015840368610158401015840E) is located 30 kmnortheast of Sofia on a 2280 ha territory while the ldquoSlivenrdquomine (42∘411015840476810158401015840N 26∘221015840224710158401015840E) is located in SouthEastern Bulgaria and occupies an area of 491 ha (Figure 1)Mining operations at the two locations were conducted in aconventional underground manner from 1962 to 1981 Theywere officially closed in 1992 and remediated until 2001

Romania

Blac

k Se

a

TurkeyGreece

Mac

edon

iaSe

rbia

-Mon

tene

gro

Bulgaria

BuhovoSofia

Sliven

Figure 1 Map of Bulgaria and the location of the studied sitesBuhovo (BuhC and BuhD) and Sliven (Sliv)

Samples from Buhovo were collected in May 2003 atdepths of 20 cm (BuhC) and 40 cm (BuhD) Samples labelledldquoSlivrdquo were collected in June 2004 from the ldquoSlivenrdquo minewaste pile at a depth of 40 cm Five samples from BuhCBuhD and Sliv were collected under sterile conditionstransported at 4∘C and stored at minus20∘C until use

22 Environmental Variables The organic matter content ofthe sample was determined by Turynrsquos method based on itsoxidation by potassium dichromate [35] The pH was mea-sured using a portable potentiometer (HANA pH meter)after the soil samples had been suspended in distilledwater (soil liquid 1 25) The concentrations of sulfatesand nitrates were determined using a spectrophotometerin 01M CaCl

2soil extract following methods described

by Bertolacini and Barney II [36] and Keeney and Nelson[37] respectively The concentration of HMs was measuredusing an ELAN 5000 Inductively Coupled Plasma MassSpectrometer (Perkin Elmer Shelton CT USA) in a 1M HClsolution (1 20 soil 1M HCl)The results were calculated foroven-dried soil

23 DNAExtraction TotalDNA (gt25 kb)was extracted fromthe samples (3 g) after direct lysis using themethod describedby Selenska-Pobell et al [38] and the DNA subsamples(five DNA subsamples for sampling site) were collected in arepresentative average sample for further analysis

24 PCR Amplification Archaeal 16S rRNA genes from thegenomic DNA were amplified via seminested PCR usingspecific archaeal 16S

21ndash40F (51015840-TTCCGGTTGATCCYGCCG-

GA-31015840) and universal 16S1492ndash1513R (51015840-ACGGYTACCTTG-

TTACGACTT-31015840) primers Each PCR reaction mixture(20120583L) contained 200 120583M deoxynucleotide triphosphates125mM MgCl

2 125mM MgCl

2 10 pmol DNA primers 1ndash

5 ng template DNA and 1U AmpliTaq Gold polymerasewith the corresponding 10x buffer (Perkin Elmer FosterCity CA USA) The amplifications were performed with aldquotouch downrdquo PCR in a thermal cycler (Biometra GottingenGermany) After an initial denaturation at 94∘C for 7minthe annealing temperature was decreased from 59 to 55∘Cover five cycles followed by 25 cycles each with a profile

Archaea 3

of denaturation at 94∘C (60 sec) 55∘C (40 sec) and 72∘C(90 sec) The amplification was completed by an extension of20min at 72∘CThe diluted products of the first reaction wereused as templates for the second round of PCR where twoarchaeal specific primers 16S

21ndash40F and 16S940ndash958R (51015840-YCC-GGCGTTGAMTCCAATT-31015840) were applied [39] The initialdenaturation at 95∘C for 7min was followed by 25 cycleseach consisting of denaturation at 94∘C (60 sec) annealingat 60∘C (60 sec) and polymerization at 72∘C (60 sec) Theamplification was completed by an extension of 10min at72∘C This seminested PCR format was applied to obtain asufficient amount of PCR products for the cloning procedure

Archaeal amoA fragments (sim635 bp)were amplified usingthe PCR primers Arch-amoAF (51015840-STAATGGTCTGGCTT-AGACG-31015840) and Arch-amoAR (51015840-GCGGCCATCCATCT-GTATGT-31015840) [40] PCR cycling was conducted according toFrancis et al [40] with an initial denaturation at 95∘C for5min followed by 35 cycles of the following denaturationat 94∘C (45 sec) annealing at 53∘C (1min) and extension at72∘C (1min) Amplificationwas completed by an extension of15min at 72∘C

25 16S rRNA Gene Clone Libraries One archaeal and oneamoAgene clone libraries for BuhC BuhD and Slivwere con-structed using the pooled products from the PCR reactionsThe 16S rDNA amplicons from five replicates were combinedand cloned directly into Escherichia coli using a TOPO TACloning Kit (Invitrogen Carlsbad CA USA) following themanufacturerrsquos instructions to generate clone libraries Thearchaeal 16S rRNA gene inserts and amoA gene insertswere subsequently amplified by PCR with plasmid-specificprimers for the vectors M13 and M13 rev and then digested(2 h 37∘C) with the MspI and HaeIII restriction enzymesfollowing the manufacturerrsquos instructions (Thermo FisherScientific USA) Restriction fragment length polymorphism(RFLP) patterns were visualized using 35 Small DNA LowMelt agarose gels (Biozym Hessisch Oldenburg Germany)and these data were then used to group clones into phylo-types The representatives of the RFLP types were purifiedusing an Edge BioSystems Quick-Step 2 PCR PurificationKit (MoBiTec Gottingen Germany) and then sequencedusing the BigDye Termination v31 Kit (Applied Biosystems)and ABI PRISM 310DNA sequencer (Applied BiosystemsFoster City CA USA) The sequencing of archaeal 16S rRNAgene fragments was performed using the primers 16S

21ndash40 Fand 16S

940ndash958R while amoA gene fragments were sequencedusing the vector primer SP6

26 Phylogenetic Analysis The sequences obtained wereanalysed and compared with those in the GenBank databaseusing the BLAST server at the National Centre for Biotech-nology Information (NCBI) (httpwwwncbinlmnihgov)The presence of chimeric sequences in the clone librarieswas determined using the programs CHIMERA CHECKavailable on the Ribosomal Database Project II (release 110)and Bellerophon [41] The sequences were aligned with thosecorresponding to the closest phylogenetic relatives using

the Clustal W program [42] Phylogenetic trees were con-structed according to the neighbour-joining method usingthe Bioedit software package

27 Data Analysis The results were statistically analysed byNCSS97 (NCSS Kaysville Utah) and the average values werepresented The sampling efficiency and diversity within thearchaeal clone libraries were estimated using the MOTHURsoftware program based on the furthest-neighbour algo-rithm and the sequences were grouped into operationaltaxonomic units (OTUs) [43] at sequence similarity levels(SSLs) of BuhC ge 97 (003 distance) BuhD ge 94 (006distance) and Sliv ge 91 (009 distance) For each samplethe archaeal OTU richness (rarefaction curves Chao 1 ACE)[44] and diversity (Shannon-Weiner index) [45] estimateswere calculated Statistical analysis of amoA OTUs was notcarried out because of the low number of unique genesequences identified in the BuhC BuhD and Sliv clonelibraries The level of pollution was expressed using a toxicityindex (TI) as follows

TI =sum119862

119894

ED50119894

(1)

where 119862119894is the concentration of metal 119894 in substratum

(mg kgminus1) and ED50 is the total concentration of metalcausing 50 reduction in microbial dehydrogenase activity(original ED50s were taken fromWelp [46])

28 Nucleotide Sequence Accession Numbers The sequencesreported in this study were deposited in GenBank under thefollowing accession numbers FM897343 to FM897356 forpartial archaeal 16S rRNA gene sequences and FM886822 toFM886831 for crenarchaeotic amoA gene sequences

3 Results

31 Environmental Variables Buhovo and Sliven samples dif-fered in their geochemistry and the levels of U and HM con-tamination BuhC and BuhD were sampled (Chromic cam-bisols) fromdifferent soil depths while Slivwas a sandy gravelmaterial collected from a mine waste pile The texture ofBuhC (20 cm at soil depth) was classified as sandy clay (35silt and 54 clay) whereas BuhD (40 cm at soil depth) wasclassified as clay (38 silt and 60 clay) The bulk densityof Buh soil varied in depth from 15-16 g cmminus3 (20 cm) to17-18 g cmminus3 (40 cm) Soil porosity was 36ndash40 (20 cm) and25ndash30 (40 cm) (personal communication)There is no dataconcerning the texture and geochemistry of Sliv substratumexcept the organic matter content (03) and pH (75) Theorganic matter content of the Buh samples was 28 forBuhC and 16 for BuhD The total amount of nitrogendecreased from 119 g kgminus1 (20 cm) to 103 g kgminus1 (40 cm)while the total amount of phosphorus was not significantlydifferent between the two soil layersmdash053 g kgminus1 (20 cm)and 051 g kgminus1 (40 cm) The pHH

2O of BuhC and BuhD was

slightly acidic (pH 69 and 66 resp)The main pollutants were Cu and Zn (BuhC BuhD and

Sliv) U (BuhC and Sliv) Cr (BuhC and BuhD) As (BuhC

4 Archaea

Table 1 Physicochemical characteristics of samples from three sites in Bulgaria polluted by uraniummining activities expressed as means plusmnstandard deviation (119899 = 15)

Parameter 119863 BC BuhC BuhD SlivpH mdash mdash 69 plusmn 03 66 plusmn 02 75 plusmn 03

OM mdash 28 plusmn 13 16 plusmn 10 03 plusmn 01

NO3-N mgkg mdash 216 plusmn 129 94 plusmn 66 199 plusmn 110

SO4 mgkg mdash 786 plusmn 950 1300 plusmn 1420 151 plusmn 140

As mgkg 384 274 plusmn 130

1

724 plusmn 28

1

412 plusmn 220

1

Cd mgkg 015 24 plusmn 13

1

11 plusmn 12 27 plusmn 18

1

Co mgkg ND 295 plusmn 12 272 plusmn 12 224 plusmn 14

Cr mgkg 5100 896 plusmn 26 952 plusmn 74 86 plusmn 19

Cu mgkg 4734 236 plusmn 114

1


1

Ni mgkg 3641 752 plusmn 134 984 plusmn 89

1

370 plusmn 110

Pb mgkg 1919 674 plusmn 394

1


1

Zn mgkg 5498 448 plusmn 520

1

464 plusmn 231

1

1270 plusmn 984

1

U mgkg 03ndash11lowast 200 plusmn 212 784 plusmn 87 374 plusmn 112

TIAs mdash mdash 163 plusmn 008 043 plusmn 002 245 plusmn 013

TICd mdash mdash 003 plusmn 001 001 plusmn 000 003 plusmn 002

TICo mdash mdash 005 plusmn 000 005 plusmn 000 004 plusmn 000

TICr mdash mdash 126 plusmn 003 134 plusmn 01 012 plusmn 002

TICu mdash mdash 674 plusmn 032 288 plusmn 060 9743 plusmn 250

TINi mdash mdash 075 plusmn 013 098 plusmn 009 037 plusmn 011

TIPb mdash mdash 103 plusmn 006 019 plusmn 002 790 plusmn 008

TIZn mdash mdash 389 plusmn 045 403 plusmn 000 1104 plusmn 086

TIsum mdash mdash 1538 991 119381Value above the maximum allowable concentration referring to Bulgarian legislation [47] lowastValues according to UNSCEAR [48] ND no data 119899 number ofsamples D dimension BC background concentrations referring to Bulgarian legislation [47] TIsum sum of toxicity indices of heavy metals (except U) andmetalloid As

and Sliv) Pb (Sliv) and sulfates (BuhD) (Table 1) All siteswere highly contaminated as shown by their individual TI

119894

(119894mdashheavy metal with TI gt 10) and TIsum which decreased asfollows Sliv (11938) gt BuhC (1538) gt BuhD (991) Moreoverthe level of toxicity might actually be stronger if the valuestook into account Mn (BuhC and BuhD) and U (BuhC andSliv) since their concentrations were also high However theTIsum did not include these due to a lack of ED50 data

32 Phylogenetic Diversity of Archaeal and amoA GeneSequences A total of 355 archaeal clones (156 from BuhC128 from BuhD and 71 from Sliv) and 229 amoA gene clones(107 from BuhC 99 from BuhD and 23 from Sliv) wereselected and their 16S rDNA inserts were analysed by RFLPThe clones sequenced were grouped into 19 (archaeal) and15 (amoA) OTUs and out of these 14OTUs and 10OTUswere unique respectively The rarefaction curves of thearchaeal BuhC (399plusmn024OTUs) BuhD (699plusmn007OTUs)and Sliv (199 plusmn 006OTUs) clone libraries were saturatedindicating that they completely covered the natural archaealdiversity of the samples and that the observed OTUs werea good representation of the archaeal community richness(Figure 2) The estimates of archaeal richness (Chao 1 ACE)and diversity (Shannon-Weiner index) predicted the highestvalues of indices in BuhD followed by the BuhC and Slivclone libraries (Table 2)

0

1

2

3

4

5

6

7

8

1 20 40 60 80 100 120 140

Num

ber O

TUs o

bser

ved

Number of sequences sampled

BuhCBuhDSliv

Figure 2 Rarefaction curves indicating archaeal 16S rRNA richnesswithin BuhC (SSL 97) BuhD (SSL 94) and Sliv (SSL 91) clonelibraries

33 Archaeal Community Composition The 16S rRNA genesequences identified in BuhC BuhD and Sliv belonged to the11bNitrososphaera cluster of Crenarchaeota (Figure 3) Rep-resentatives of other crenarchaeotic clades or other archaealphyla were not detected in this study

Archaea 5

Table 2 Predicted richness (Chao 1 and ACE) and diversity (Shannon-Weiner index) of BuhC BuhD and Sliv 16S rDNA archaeal clonelibraries expressed as means plusmn standard deviation

Clone library Number of clones Number of OTUs Number of singletonsdoubletons Chao 1 ACE Shannon-Weiner indexBuhCa 156 7 4 4 plusmn 025 NA 097 plusmn 010

BuhDb 128 8 1 7 plusmn 000 7 plusmn 000 151 plusmn 013

Slivc 71 3 1 2 plusmn 000 2 plusmn 000 032 plusmn 024

OTUs were defined at a3 b6 and c9 differences in 16S rRNA gene sequences

01

Acidianus ambivalens DS3772 (D85506)Pyrobaculum islandicum geo2 (L07511)

BuhC-Ar48 (FM897345) 37 clonesBuhD-Ar9 clonesSLA-AM3-1 (JQ978502 permafrost soil) OUT-G3-5 (JQ668646 oil reservoir) BuhD-Ar111 (FM897353) 5 clonesBuhC-Ar7 clones660mArA8 (AY367312 water depth borehole)

QA4 (FJ790596 quartz in a Tibet desert)Sliv-Ar22 (FM897354) 40 clones

TX1G10 (FJ784315 alkaline soil)

Sliv-Ar32 (FM897355) 30 clonesGitt-GR-31 (AJ535119 uranium mine waste)Gitt-GR-39 (AJ535120 uranium mine waste)

KAVG11AR3 (JN863130 iron-ore mine rhizosphere)BuhD-Ar100 (FM897352) 15 clonesM26-6Ar07 (HM998417 deep-sea sediment)

UMV3A164 (HM584831 mud volcano)BuhC-Ar44 (FM897347) 1 cloneBuhD-Ar15 clones

BuhD-Ar5 (FM897350) 6 clonesW5P2-D12 (GQ871411 agricultural soil)LIM-A88 (JF737830 limestone rock)

BuhC-Ar18 (FM897344) 38 clonesBuhD-Ar14 clonesBuhC-Ar33 (FM897346) 1 cloneTP-SL-A-12 (HQ738979 permafrost soil)BAVG11AR21 (JQ668088 iron-ore mine soil rhizosphere)BuhC-Ar58 (FM897348) 1 cloneTX1C03 (FJ784296 alkaline soil)54D9 (AY278106 terrestrial)Sliv-Ar44 (FM897356) 1 clone

BuhC-Ar8 (FM897343) 70 clonesBuhD-Ar62 clonesBuhC-Ar67 (FM897349) 1 clone

Gitt-GR-74 (AJ535122 uranium mine waste)BuhD-Ar78 (FM897351) 2 clones

TP-SL-A-28 (HQ738987 permafrost soil)SCA1154 (U62814 agricultural soil)

Clus

ter A

Clus

ter B

I

II

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster

K09 0 56 (AB541694 soil cattle manure compost)

arcBiof 0314 (KC604547 pristine aquifer)

Candidatus Nitrososphaera gargensis Ga92 (NR 102916)

Figure 3 Phylogenetic analysis of archaeal 16S rRNA gene sequences retrieved from uranium mining sites BuhC BuhD and Sliv The treewas constructed using the neighbour-joiningmethodThe 16S rRNA sequences ofAcidianus ambivalensDS3772 and Pyrobaculum islandicumgeo2 were used as an outgroup The scale bar represents 01 changes per nucleotide position

6 Archaea

01

Nitrospira briensis (U76553)

LZT1-A58 (GQ226128 hot spring)

Sliv-A-30 (FM886831) 6 clones


BuhD-A-3 (FM886824) 2 clonesBuhC-A1 clone

SF05-BA10-G01 (EU651210 estuary sediment)

PP-E1 (JQ638739 soil)

S-A1 (JF935924 bulk soil)

BuhC-A-18 (FM886822) 3 clones

BuhD-A-115 (FM886829) 14 clones


AOA-OTU4 (HQ267736 grassland soil)

L-A2 (JF935852 bulk soil)

BuhD-A-66 (FM886826) 8 clonesBuhC-A 4 clones

GSWuWeiaoa-44 (FN691264 arable soil)

BuhD-A-80 (FM886827) 1 clone

136 (HQ007844 vegetated soil)

TH083269-4-80UL-9 (JQ277528 wastewater treatment plant)


P2-40 (HM803786 arable soil)

AOA-8 (JF735056 soil of plateau wetland)

BuhD-A-24 (FM886825) 55 clonesBuhC-A92 clones

Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster

LSbf AOA 43 (HQ401433 freshwater flow channel)LSbf AOA 10 (HQ401411 freshwater flow channel)

4F 4 (EU671839 grassland soil)

LNbf AOA 47 (HQ401473 freshwater flow channel)

AM 2 (HQ317053 wastewater treatment plant)

LSbf AOA 41 (HQ401432 freshwater flow channel)

AS amoA-OUT-3-3 (HQ221889 Ammerbach stream)

Figure 4 Phylogenetic analysis of archaeal amoA gene sequences retrieved from uranium mining sites BuhC BuhD and Sliv The tree wasconstructed using the neighbour-joiningmethodThe amoA sequence ofNitrospira briensiswas used as an outgroupThe scale bar represents01 changes per nucleotide position

The crenarchaeotic sequences were grouped into clusters(A and B Figure 3) Cluster A involved 16S rRNA genesequences retrieved mainly from the highly polluted envi-ronments of Sliv and BuhC Cluster B consisted of OTUsfrom the BuhC and BuhD (226 of 227 clones) libraries Thelatter cluster was separated into subcluster IB generated bythe sequences of the BuhD clone library (36 of 37 clones) andsubcluster IIB which mainly consisted of clones belonging tothe BuhC and BuhD libraries (190 of 196 clones)

There were common (BuhC-Ar8 BuhC-Ar18 BuhC-Ar48 and BuhD-Ar111) 16S rRNA gene archaeal sequences inthe clone libraries of BuhC andBuhDWe did not retrieve anygene sequences common to the Sliv and Buh substrata

All retrieved 16S rRNA gene sequences matched tosequences of uncultured archaea except Sliv-Ar32 which

was affiliated with the cultured archaeon Candidatus Nitro-sosphaera gargensis (NR 102916)

34 Composition of the amoA Community Phylogeneticanalysis of 10 archaeal amoA OTUs revealed a high sequenceidentity (98ndash100) with ammonia-oxidizing crenarchaeotesCluster I from the phylogenetic tree of the amoA genesequences was formed by two OTUs from Sliv whereas clus-ters II and III were only composed of OTUs from the Buhovosoil environments (Figure 4) In total all amoA OTUs werepresented in a relatively small number of clones (1ndash15 clones)except BuhD-A-24 and its analogue OTU from BuhC whichconsisted of 55 and 92 clones respectively

All retrieved archaeal amoA sequences were matchedwith uncultured crenarchaeotes

Archaea 7

Protein sequences derived from the same samples werealso analysed and the data validated our DNA results(data not published) The protein sequences exhibited 96ndash100 similarity to the closest matched GenBank sequencesretrieved from terrestrial estuarine and hot spring environ-ments

4 Discussion

The BuhC BuhD and Sliv archaeal communities appearto be composed solely of members of the soil-freshwater-subsurface group (11b) of Crenarchaeota which was recentlyassigned by Bartossek et al [49] asNitrososphaera clusterThepresence of Crenarchaeota in these sites was not surprisingsince these organisms are widespread [4 7 50] even inenvironments highly polluted with U and HMs [5 7 13 51]Probably the selection and propagation of only 11b Crenar-chaeota in Buhovo and Sliven are passed under the power ofU and HM pollution Supporting this notion Geissler et al[52] Reitz et al [14] and Radeva et al [53] reported a strongreduction in archaeal diversity and a shift from Crenarchae-ota 11a to 11b in soil samples supplemented with uranylnitrateThe adverse effects of U were also confirmed by Poratet al [5] who found low archaeal diversity in U-nitrate-contaminated sediments of theOak Ridge stream (TNUSA)

The importance of the substratum and the level of pollu-tion in the pattern of crenarchaeotic distribution is evidentfrom the archaeal phylogenetic tree (Figure 3) where OTUsare grouped in one large cluster (B) based on 16S rRNA genesequences from Buhovo soil (9 of 10OTUs226 of 227 clones)and another smaller cluster (A) formed of OTUs from themost polluted environments Sliv and BuhC (4 of 6OTUs114 of 128 clones) There are no common 16S rRNA genesequences from the two substrata (Buh soil and Sliv sandygravel matter) studied

The distinct physical and geochemical niches of thesites harbour characteristic crenarchaeotic populations (Fig-ure 3) (i) typical soil species tolerant towards environmentalextremes including resistance to U and HMs (members ofsubcluster IIB) (ii) depth specific species probably sensitiveto U and HMs (members of subcluster IB) and (iii) resistanttoU andHM soil and rocky inhabitants (cluster A) All OTUscorrespond to terrestrial environmental matches except Sliv-Ar44 BuhD-Ar100 and BuhD-Ar111 which exhibit highsimilarity (99-100) with gene sequences derived fromaquatic environments groundwater (KC604547) deep-seasediments (HM998417) and seawater at depths of 660m(AY367312) respectively In general the above-mentionedwater-related OTUs are only represented by a small numberof clones (1ndash15)

The Buh soil environments comprise more complex andmore diverse archaeal communities 84 of OTUs and 80of archaeal clones are from Buh which validates data fromOchsenreiter et al [7] indicating that the 11b crenarchaeoticclade is a typical ldquosoil lineagerdquo

Archaeal diversity in Buh soil is relatively low varyingfrom 097 (BuhC) to 151 (BuhD) and is depth depen-dent Archaeal communities of the two soil depths include

both common (BuhC-Ar8 BuhC-Ar18 BuhC-Ar44 BuhC-Ar48 and BuhD-Ar111) and depth-specific 16S rRNA genesequences the latter of which are represented by a smallnumber of clones (1ndash15 clones) The dominant OTU BuhC-Ar8 is equally distributed in soil depth comprising 45 and48 of clones retrieved from BuhC and BuhD respectivelyMoreover it is closely affiliated (99 SSL) with the uncul-tured crenarchaeote Gitt-GR-74 (AJ535122) which is foundin uranium mill tailing in Saxony Germany [13]

A trend for depth dependency in archaeal distributionwas also observed in other studies which indicate thatCrenarchaeota are more abundant in deeper soil layers [54ndash57] and that archaeal bacterial ratios increase with soil depth[2] In the aforementioned studies increasing abundance ofcrenarchaeotes correlated with decreasing nutrient (organiccarbon and inorganic nitrogen) and oxygen concentrationsin deeper soil layers In agreement with the above-mentionedstatements we can speculate for BuhD that the diversityof Crenarchaeota is favoured by the nutritional and oxygenstatus of this soil depth and its low levels of U and HMpollution The relative opposite conditions in BuhC soillayer comparing to BuhD (higher organic matter contenthigher aeration in the upper soil layer and higher levelsof U and HMs) limit its archaeal diversity mainly to threedominant OTUs (BuhC-Ar8 BuhC-Ar18 and BuhC-Ar48)that harboured 93 of clones in the BuhC clone library

The sandy gravel substratum of Sliv and its high levelof pollution make this environment very unfavourable forarchaeal proliferation The inhabitants of Sliv are presentedby two main OTUs (Sliv-Ar32 and Sliv-Ar22) that com-prise 99 of clones All archaeal 16S rRNA gene sequencesretrieved from Sliv correspond with uncultured crenar-chaeotic matches except Sliv-Ar32 which exhibits a 99similarity with Candidatus Nitrososphaera gargensis Ga92According to Spang et al [58]Ca N gargensis is well adaptedto HM-contaminated environments and encodes a numberof HM resistance genes that convey the genetic capacity torespond to environmental changes The close similarity ofSliv-Ar32 to Gitt-GR sequences (99 SSL) recovered fromU mill tailings in Germany also confirms the high toleranceof Sliv-Ar32 towards U and HM pollution The other moreabundant OTU is Sliv-Ar22 (40 clones) and its dominance inSliv clone library can be explained by both tolerance towardshigh levels of pollution and ability of Sliv-Ar22 archaeonto colonize rocky substrata This sequence exhibits highsimilarity to the uncultured crenarchaeote QA4 (99 SSL)which was recovered from quartz rocks located in the high-altitude tundra of Central Tibet [59]

The phylogenetic analysis of archaeal amoA genesequences retrieved from BuhC BuhD and Sliv revealsthat the Crenarchaeota inhabiting these locations harbourammonia oxidizers (Figure 4) The pattern of amoA genesequence distribution is similar to that of Crenarchaeotawith the smallest number of OTUs in the most unfavourableenvironment of Sliv (2OTUs23 clones) followed by thehighly polluted BuhC (5OTUs107 clones) and the relativelylow polluted BuhD (6OTUs99 clones) The high numberof amoA OTUs in BuhD is related to the highest archaealdiversity in this depth and is due to the favourable conditions

8 Archaea

(low organic matter nitrogen and oxygen content and highclayey soil texture) which stimulate not only the archaealdiversity but also the diversity of ammonia-oxidizingarchaea To date studies [33 60ndash63] that have investigatedthe environmental factors that shape amoA gene diversityin oceans sediments and soils have identified these factorsas key environmental parameters for the proliferation ofammonia-oxidizing archaea

Forty-six percent of the archaeal amoA OTUs whichcomprise 73 of clones retrieved in this study affiliate witharchaeal amoA gene sequences obtained from freshwaterecosystems [64 65] and wastewater treatment plants [66]These belong to the ldquosoil and other environmentsrdquo clusteras proposed by Prosser and Nicol [67] The other amoAOTUs (all from BuhD and BuhC) exhibit gene sequencesclosely related to those retrieved from soil environments likebulk [60] and arable (FN691264 HM803786) soils grassland(HQ267736 EU671839) and semiarid soil (JQ638739) thatbelong also to the ldquosoil and other environmentsrdquo cluster [67]

BuhC and BuhD are very different environments withregard to soil texture nutrients oxygen (low soil porosity)and pollution status Nevertheless the two environments areinhabited by ammonia-oxidizing archaea as determined bythe presence of the amoA gene sequence BuhD-A-24 com-prised 23 (BuhD) and 41 (BuhC) of all retrieved amoAclones It is likely that the exclusive domination of BuhD-A-24 in Buhovo soil depths is a result of the adverse effectsof pollution that reduce archaeal amoA diversity and theselection of only a few resistant gene sequences We did notdetect novel archaeal amoA clusters that would indicate theexistence of special U- andHM-resistant ammonia-oxidizingarchaea in the sites studied This reveals the widespreaddistribution of ammonia-oxidizing archaea and the capacityof some species to tolerate high levels of U and HMs

5 Conclusions

Phylogenetic analysis revealed that all archaeal 16S rRNAgene sequences assessed in this study belong to the 11bNitrososphaera cluster of CrenarchaeotaThe diversity of cre-narchaeotic communities that inhabit the three sites of inter-est was very low especially in the high U- and HM-pollutedsandy-stone environment of the Sliv mineThe archaeal com-munities of Buh and Sliv mines were distinct to each site anddid not harbour common gene sequences We did not detectnovel crenarchaeotic and amoA gene clusters indicating thatthe polluted environments of Buh and Sliv are inhabited bytypical archaeal soil lineages It is likely that these archaealsoil lineages were selected by the multifactorial nature of thelocal environment resulting in the development of toleranceof indigenous archaea to high U and HM pollution Thearchaeal amoA gene sequences detected in BuhC BuhD andSliv supposed that ammonia-oxidizing archaea participate innitrogen cycling in environments highly polluted with U andHMsThis studywill be helpful in understanding the archaealand ammonia-oxidizing archaeal diversities in soils pollutedwith U and HMs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This study was financially supported by the Institute ofResource Ecology Helmholtz-Centre Dresden-RossendorfGermany

References

[1] D R Rogers and K L Casciotti ldquoAbundance and diversity ofarchaeal ammonia oxidizers in a coastal groundwater systemrdquoApplied and Environmental Microbiology vol 76 no 24 pp7938ndash7948 2010

[2] D Kemnitz S Kolb and R Conrad ldquoHigh abundance of Cre-narchaeota in a temperate acidic forest soilrdquo FEMSMicrobiologyEcology vol 60 no 3 pp 442ndash448 2007

[3] E Teira P Lebaron H van Aken and G J Herndl ldquoDistri-bution and activity of Bacteria and Archaea in the deep watermasses of the North Atlanticrdquo Limnology and Oceanographyvol 51 no 5 pp 2131ndash2144 2006

[4] C Schleper G Jurgens andM Jonuscheit ldquoGenomic studies ofuncultivated Archaeardquo Nature Reviews Microbiology vol 3 no6 pp 479ndash488 2005

[5] I Porat T A Vishnivetskaya J J Mosher et al ldquoCharacteriza-tion of archaeal community in contaminated and uncontami-nated surface stream sedimentsrdquoMicrobial Ecology vol 60 no4 pp 784ndash795 2010

[6] K Zhalnina P Dorr de Quadros F A O Camargo and E WTriplett ldquoDrivers of archaeal ammonia-oxidizing communitiesin soilrdquo Frontiers in Microbiology vol 3 article 210 2012

[7] T Ochsenreiter D Selezi A Quaiser L Bonch-Osmolovskayaand C Schleper ldquoDiversity and abundance of Crenarchaeota interrestrial habitats studied by 16S RNA surveys and real timePCRrdquo Environmental Microbiology vol 5 no 9 pp 787ndash7972003

[8] A Geissler Prokaryotic microorganisms in uranium miningwaste piles and their interactions with uranium and other heavymetals [PhD thesis] TU Bergakademie Freiberg FreibergGermany 2007

[9] T Reitz M L Merroun A Rossberg and S Selenska-PobellldquoInteractions of Sulfolobus acidocaldarius with uraniumrdquo Radi-ochimica Acta vol 98 no 5 pp 249ndash257 2010

[10] K Takai D P Moser M DeFlaun T C Onstott and J K Fred-rickson ldquoArchaeal diversity in waters from deep South Africangold minesrdquo Applied and Environmental Microbiology vol 67no 12 pp 5750ndash5760 2001

[11] L Y Stein G Jones B Alexander K Elmund C Wright-Jonesand K H Nealson ldquoIntriguing microbial diversity associatedwith metal-rich particles from a freshwater reservoirrdquo FEMSMicrobiology Ecology vol 42 no 3 pp 431ndash440 2002

[12] Y Suzuki S D Kelly K M Kemner and J F Banfield ldquoDirectmicrobial reduction and subsequent preservation of uraniumin natural near-surface sedimentrdquo Applied and EnvironmentalMicrobiology vol 71 no 4 pp 1790ndash1797 2005

[13] G Radeva and S Selenska-Pobell ldquoArchaeal diversity in soilsof the uranium mining wastesrdquo Annual Report of Institute ofRadiochemistry FZR-373 2002

Archaea 9

[14] T Reitz A Geissler and S Selenska-Pobell ldquoChanges inarchaeal community of the waste pile Haberland induced byuranyl nitrate treatmentsrdquo Annual Report of the Institute ofRadiochemistry FZR-459 2006

[15] K Kashefi E S Shelobolina W C Elliott and D R Lov-ley ldquoGrowth of thermophilic and hyperthermophilic Fe(III)-reducing microorganisms on a ferruginous smectite as the soleelectron acceptorrdquo Applied and Environmental Microbiologyvol 74 no 1 pp 251ndash258 2008

[16] A J Francis J B Gillow C J Dodge R Harris T J Beveridgeand H W Papenguth ldquoUranium association with halophilicand non-halophilic Bacteria and Archaeardquo Radiochimica Actavol 92 no 8 pp 481ndash488 2004

[17] T Reitz M L Merroun A Rossberg R Steudtner and SSelenska-Pobell ldquoBioaccumulation of U(VI) by Sulfolobus aci-docaldarius under moderate acidic conditionsrdquo RadiochimicaActa vol 99 no 9 pp 543ndash553 2011

[18] M Pester C Schleper and M Wagner ldquoThe Thaumarchaeotaan emerging view of their phylogeny and ecophysiologyrdquo Cur-rent Opinion in Microbiology vol 14 no 3 pp 300ndash306 2011

[19] M Konneke A E Bernhard J R de la Torre C B WalkerJ B Waterbury and D A Stahl ldquoIsolation of an autotrophicammonia-oxidizing marine archaeonrdquo Nature vol 437 no7058 pp 543ndash546 2005

[20] A H Treusch S Leininger A Kietzin S C Schuster H-PKlenk and C Schleper ldquoNovel genes for nitrite reductase andAmo-related proteins indicate a role of uncultivatedmesophilicCrenarchaeota in nitrogen cyclingrdquo Environmental Microbiol-ogy vol 7 no 12 pp 1985ndash1995 2005

[21] S Leininger T Urich M Schloter et al ldquoArchaea predominateamong ammonia-oxidizing prokaryotes in soilsrdquo Nature vol442 no 7104 pp 806ndash809 2006

[22] M J L Coolen B Abbas J van Bleijswijk et al ldquoPutativeammonia-oxidizing Crenarchaeota in suboxic waters of theBlack Sea a basin-wide ecological study using 16S ribosomaland functional genes and membrane lipidsrdquo EnvironmentalMicrobiology vol 9 no 4 pp 1001ndash1016 2007

[23] P LamMM Jensen G Lavik et al ldquoLinking crenarchaeal andbacterial nitrification to anammox in the Black Seardquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 17 pp 7104ndash7109 2007

[24] C Wuchter B Abbas M J L Coolen et al ldquoArchaeal nitri-fication in the oceanrdquo Proceedings of the National Academy ofSciences of the United States of America vol 103 no 33 pp12317ndash12322 2006

[25] M Herrmann A M Saunders and A Schramm ldquoArchaeadominate the ammonia-oxidizing community in the rhizo-sphere of the freshwater macrophyte Littorella uniflorardquoAppliedand Environmental Microbiology vol 74 no 10 pp 3279ndash32832008

[26] J R de la Torre C B Walker A E Ingalls M Konneke andD A Stahl ldquoCultivation of a thermophilic ammonia oxidizingarchaeon synthesizing crenarchaeolrdquo Environmental Microbiol-ogy vol 10 no 3 pp 810ndash818 2008

[27] R Hatzenpichler E V Lebedeva E Spieck et al ldquoA moderatelythermophilic ammonia-oxidizing crenarchaeote from a hotspringrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 105 no 6 pp 2134ndash2139 2008

[28] L J Reigstad A Richter H Daims T Urich L Schwark andC Schleper ldquoNitrification in terrestrial hot springs of Icelandand Kamchatkardquo FEMSMicrobiology Ecology vol 64 no 2 pp167ndash174 2008

[29] J Pouliot P E Galand C Lovejoy and W F Vincent ldquoVerticalstructure of archaeal communities and the distribution ofammonia monooxygenase A gene variants in two meromicticHigh Arctic lakesrdquo Environmental Microbiology vol 11 no 3pp 687ndash699 2009

[30] P W J J van der Wielen S Voost and D van der KooijldquoAmmonia-oxidizing Bacteria and Archaea in groundwatertreatment and drinking water distribution systemsrdquo Appliedand Environmental Microbiology vol 75 no 14 pp 4687ndash46952009

[31] H-D Park G F Wells H Bae C S Griddle and C A FrancisldquoOccurrence of ammonia-oxidizing Archaea in wastewatertreatment plant bioreactorsrdquoApplied and Environmental Micro-biology vol 72 no 8 pp 5643ndash5647 2006

[32] G W Nicol S Leininger C Schleper and J I Prosser ldquoTheinfluence of soil pH on the diversity abundance and transcrip-tional activity of ammonia oxidizing Archaea and BacteriardquoEnvironmentalMicrobiology vol 10 no 11 pp 2966ndash2978 2008

[33] K L Adair and E Schwartz ldquoEvidence that ammonia-oxidizingArchaea are more abundant than ammonia-oxidizing Bacteriain semiarid soils of Northern Arizona USArdquoMicrobial Ecologyvol 56 no 3 pp 420ndash426 2008

[34] L-M Zhang P R Offre J-Z He D T Verhamme G WNicol and J I Prosser ldquoAutotrophic ammonia oxidation by soilthaumarchaeardquo Proceedings of the National Academy of Sciencesof the United States of America vol 107 no 40 pp 17240ndash172452010

[35] I S Kaurichev ldquoOrganic matter determination in soil samplesbyThurinrsquosmethodrdquo inManual of Pedological Practices pp 212ndash241 Kolos Moscow Russia 1980

[36] R J Bertolacini and J E Barney II ldquoColorimetric determina-tion of sulfate with barium chloranilaterdquo Analytical Chemistryvol 29 no 2 pp 281ndash283 1957

[37] D R Keeney and D W Nelson ldquoNitrogen-inorganic formsrdquo inMethods of Soil Analysis Part 2 A L Page R H Miller and DKeeney Eds vol 9 ofAgronomyMonograph pp 643ndash698 ASAand SSSA Madison Wis USA 2nd edition 1982

[38] S Selenska-Pobell G Kampf K Flemming G Radeva and GSatchanska ldquoBacterial diversity in soil samples from two ura-nium waste piles as determined by rep-APD RISA and 16SrDNA retrievalrdquo Antonie van Leeuwenhoek vol 79 no 2 pp149ndash161 2001

[39] E F DeLong ldquoArchaea in coastal marine environmentsrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 89 no 12 pp 5685ndash5689 1992

[40] C A Francis K J Roberts J M Beman A E Santoro and B BOakley ldquoUbiquity and diversity of ammonia-oxidizing Archaeain water columns and sediments of the oceanrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 102 no 41 pp 14683ndash14688 2005

[41] T Huber G Faulkner and P Hugenholtz ldquoBellerophon a pro-gram to detect chimeric sequences in multiple sequence align-mentsrdquo Bioinformatics vol 20 no 14 pp 2317ndash2319 2004

[42] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

10 Archaea

[43] P D Schloss S L Westcott T Ryabin et al ldquoIntroducingmothur open-source platform-independent community-sup-ported software for describing and comparing microbial com-munitiesrdquo Applied and Environmental Microbiology vol 75 no23 pp 7537ndash7541 2009

[44] A Chao ldquoNonparametric estimation of the number of classesin a populationrdquo Scandinavian Journal of Statistics vol 11 pp265ndash270 1984

[45] A E Magurran Ecological Diversity and Its MeasurementsPrinceton University Press Princeton NJ USA 1988

[46] G Welp ldquoInhibitory effects of the total and water-solubleconcentrations of nine different metals on the dehydrogenaseactivity of a loess soilrdquo Biology and Fertility of Soils vol 30 no1-2 pp 132ndash139 1999

[47] ldquoBulgarian legislation Ordinance 31 08rdquo Ministry of Envi-ronment andWater 2008 httpwww3moewgovernmentbgshow=topampcid=388

[48] UNSCEARmdashUnited Nations Scientific Committee on theEffects of Atomic Radiation Sources and Effects of Ionizing Radi-ation 1993

[49] R Bartossek A Spang G Weidler A Lanzen and C SchleperldquoMetagenomic analysis of ammonia-oxidizing Archaea affili-ated with the soil grouprdquo Frontiers inMicrobiology vol 3 article208 2012

[50] S T Bates D Berg-Lyons J G Caporaso W A Walters RKnight and N Fierer ldquoExamining the global distribution ofdominant archaeal populations in soilrdquo ISME Journal vol 5 no5 pp 908ndash917 2011

[51] G W Weidler M Dornmayr-Pfaffenhuemer F W Gerbl WHeinen and H Stan-Lotter ldquoCommunities of Archaea andBacteria in a subsurface radioactive thermal spring in the Aus-trian central alps and evidence of ammonia-oxidizing Crenar-chaeotardquoApplied and Environmental Microbiology vol 73 no 1pp 259ndash270 2007

[52] A Geissler T Reitz J Tschikov and S Selenska-Pobell ldquoInflu-ence of U (VI) and nitrate on microbial communities ofuranium mining wasterdquo Geophysical Research Abstracts vol 8Article ID 04336 2006

[53] G Radeva V Buchvarova K Flemming T Reitz and SSelenska-Pobell ldquoMicrobial diversity in highly contaminateduranium mining wastes Part A archaeal diversityrdquo AnnualReport of Institute of Radiochemistry FZR-511 2008

[54] C M Hansel S Fendorf P M Jardine and C A FrancisldquoChanges in bacterial and archaeal community structure andfunctional diversity along a geochemically variable soil profilerdquoApplied and Environmental Microbiology vol 74 no 5 pp1620ndash1633 2008

[55] M Hartmann S Lee S J Hallam andWW Mohn ldquoBacterialarchaeal and eukaryal community structures throughout soilhorizons of harvested and naturally disturbed forest standsrdquoEnvironmentalMicrobiology vol 11 no 12 pp 3045ndash3062 2009

[56] K G Eilers S Debenport S Anderson and N Fierer ldquoDiggingdeeper to find unique microbial communities the strong effectof depth on the structure of bacterial and archaeal communitiesin soilrdquo Soil Biology and Biochemistry vol 50 pp 58ndash65 2012

[57] A E Santoro and K L Casciotti ldquoEnrichment and character-ization of ammonia-oxidizing Archaea from the open oceanphylogeny physiology and stable isotope fractionationrdquo ISMEJournal vol 5 no 11 pp 1796ndash1808 2011

[58] A Spang A Poehlein P Offre et al ldquoThe genome ofthe ammonia-oxidizing Candidatus Nitrososphaera gargensis

insights into metabolic versatility an environmental adapta-tionsrdquoEnvironmentalMicrobiology vol 14 no 12 pp 3122ndash31452012

[59] F K Y Wong D C Lacap M C Y Lau J C Aitchison D ACowan and S B Pointing ldquoHypolithic microbial communityof quartz pavement in the high-altitude tundra of central tibetrdquoMicrobial Ecology vol 60 no 4 pp 730ndash739 2010

[60] MC Pereira e Silva F PolyNGuillaumaud J D vanElsas andJ F Salles ldquoFluctuations in ammonia oxidizing communitiesacross agricultural soils are driven by soil structure and pHrdquoFrontiers in Microbiology vol 3 article 77 2012

[61] A C Mosier and C A Francis ldquoRelative abundance and diver-sity of ammonia-oxidizing Archaea and Bacteria in the SanFrancisco Bay estuaryrdquo Environmental Microbiology vol 10 no11 pp 3002ndash3016 2008

[62] J-P Shen L-M Zhang Y-G Zhu J-B Zhang and J-Z HeldquoAbundance and composition of ammonia-oxidizing Bacteriaand ammonia-oxidizing Archaea communities of an alkalinesandy loamrdquo Environmental Microbiology vol 10 no 6 pp1601ndash1611 2008

[63] J Ollivier W Natasia A Austruy et al ldquoAbundance and diver-sity of ammonia oxidizing prokaryotes in the root-rhizospherecomplex of Miscanthus x giganteus grown in heavy metal-contaminated soilsrdquoMicrobial Ecology vol 64 no 4 pp 1038ndash1046 2012

[64] M Herrmann A Scheibe S Avrahami and K Kusel ldquoAmmo-nium availability affects the ratio of ammonia-oxidizing Bacte-ria to ammonia-oxidizing Archaea in simulated creek ecosys-temsrdquo Applied and Environmental Microbiology vol 77 no 5pp 1896ndash1899 2011

[65] H Jiang Q Huang H Dong et al ldquoRNA-based investigation ofammonia-oxidizingArchaea in hot springs of Yunnan ProvinceChinardquoApplied and Environmental Microbiology vol 76 no 13pp 4538ndash4541 2010

[66] M Muszligmann I Brito A Pitcher et al ldquoThaumarchaeotesabundant in refinery nitrifying sludges express amoA but arenot obligate autotrophic ammonia oxidizersrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 108 no 40 pp 16771ndash16776 2011

[67] J I Prosser and G W Nicol ldquoRelative contributions of Archaeaand Bacteria to aerobic ammonia oxidation in the environ-mentrdquoEnvironmentalMicrobiology vol 10 no 11 pp 2931ndash29412008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Archaea

To date little is known concerning the interactionsbetween archaea and U or HMs Kashefi et al [15] pub-lished that the hyperthermophilic crenarchaeote Pyrobacu-lum islandicum is able to reduce U(VI) to U(IV) under anaer-obic conditions at 100∘C Francis et al [16] demonstratedthat the halophilic euryarchaeote Halobacterium halobiumaccumulates high amounts of U(VI) as extracellular uranylphosphate deposits however these two organisms are notfound in U-contaminated substrata Later Reitz et al [9 17]revealed the capacity of the acidothermophilic Sulfolobusacidocaldarius which is an indigenous archaeon for U-contaminated soils and mine tailings to accumulated intra-cellular U(VI)

Thediscovery that somemesophilic archaea fromCrenar-chaeota which were later categorized into the newThaumar-chaeota phylum [18] have the potential to oxidize ammoniasuggests an important role of archaea in the nitrogen (N)cycle [19 20] The crenarchaeotic ammonia monooxygenasegene (amoA) is found in many natural environments suchas soil [2 21] marine and freshwater ecosystems [22ndash25]several geothermal environments and hot springs [26ndash28]Artic lakes [29] drinking water production plants [30] andwastewater treatment plants [31] This widespread distri-bution indicates the ubiquity and significance of archaealammonia oxidizers in the global N cycle [21 32ndash34] How-ever there are few studies assessing the abundance of archaealamoA and its diversity in U-impacted environments

Intensive U mining and milling in Bulgaria were per-formed between 1946 and 1990 and have caused significantsoil and water pollution U production was stopped by agovernment decree in 1992 andmines and tailings were tech-nically liquidated and gradually remediated Neverthelesstheir surroundings are still highly contaminated and furthercontamination from the compromised remediation of minesand tailings has been recorded

The aim of this study was to investigate the diversity ofarchaeal communities inhabiting environments impacted byU mining and milling activities and in particular to revealthe diversity of the archaeal amoA gene Since U and HMcontamination represent an old environmental burden weexpected that the composition and diversity of archaeal andamoA communities were stabilized under the selective powerof both contamination level and environmental characteris-tics

2 Materials and Methods

21 Sites and Sampling Two locations in Bulgaria were stud-ied the abandoned mining and milling complex ldquoBuhovordquoand the ldquoSlivenrdquo mine both of which have been classified asareas of high radiological risk by the Bulgarian Agency forRadiobiology and Radioprotection The mining complexldquoBuhovordquo (42∘451015840512010158401015840N 23∘341015840368610158401015840E) is located 30 kmnortheast of Sofia on a 2280 ha territory while the ldquoSlivenrdquomine (42∘411015840476810158401015840N 26∘221015840224710158401015840E) is located in SouthEastern Bulgaria and occupies an area of 491 ha (Figure 1)Mining operations at the two locations were conducted in aconventional underground manner from 1962 to 1981 Theywere officially closed in 1992 and remediated until 2001

Romania

Blac

k Se

a

TurkeyGreece

Mac

edon

iaSe

rbia

-Mon

tene

gro

Bulgaria

BuhovoSofia

Sliven

Figure 1 Map of Bulgaria and the location of the studied sitesBuhovo (BuhC and BuhD) and Sliven (Sliv)

Samples from Buhovo were collected in May 2003 atdepths of 20 cm (BuhC) and 40 cm (BuhD) Samples labelledldquoSlivrdquo were collected in June 2004 from the ldquoSlivenrdquo minewaste pile at a depth of 40 cm Five samples from BuhCBuhD and Sliv were collected under sterile conditionstransported at 4∘C and stored at minus20∘C until use

22 Environmental Variables The organic matter content ofthe sample was determined by Turynrsquos method based on itsoxidation by potassium dichromate [35] The pH was mea-sured using a portable potentiometer (HANA pH meter)after the soil samples had been suspended in distilledwater (soil liquid 1 25) The concentrations of sulfatesand nitrates were determined using a spectrophotometerin 01M CaCl

2soil extract following methods described

by Bertolacini and Barney II [36] and Keeney and Nelson[37] respectively The concentration of HMs was measuredusing an ELAN 5000 Inductively Coupled Plasma MassSpectrometer (Perkin Elmer Shelton CT USA) in a 1M HClsolution (1 20 soil 1M HCl)The results were calculated foroven-dried soil

23 DNAExtraction TotalDNA (gt25 kb)was extracted fromthe samples (3 g) after direct lysis using themethod describedby Selenska-Pobell et al [38] and the DNA subsamples(five DNA subsamples for sampling site) were collected in arepresentative average sample for further analysis

24 PCR Amplification Archaeal 16S rRNA genes from thegenomic DNA were amplified via seminested PCR usingspecific archaeal 16S

21ndash40F (51015840-TTCCGGTTGATCCYGCCG-

GA-31015840) and universal 16S1492ndash1513R (51015840-ACGGYTACCTTG-

TTACGACTT-31015840) primers Each PCR reaction mixture(20120583L) contained 200 120583M deoxynucleotide triphosphates125mM MgCl

2 125mM MgCl

2 10 pmol DNA primers 1ndash

5 ng template DNA and 1U AmpliTaq Gold polymerasewith the corresponding 10x buffer (Perkin Elmer FosterCity CA USA) The amplifications were performed with aldquotouch downrdquo PCR in a thermal cycler (Biometra GottingenGermany) After an initial denaturation at 94∘C for 7minthe annealing temperature was decreased from 59 to 55∘Cover five cycles followed by 25 cycles each with a profile

Archaea 3





21ndash40 Fand 16S





TI =sum119862

119894

ED50119894

(1)




3 Results





4 Archaea







1

724 plusmn 28

1

412 plusmn 220

1


1


1




1


1


1

370 plusmn 110


1


1


1

464 plusmn 231

1

1270 plusmn 984

1












119894



0

1

2

3

4

5

6

7

8

1 20 40 60 80 100 120 140

Num

ber O

TUs o

bser

ved


BuhCBuhDSliv



Archaea 5






01













Clus

ter A

Clus

ter B

I

II

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster





6 Archaea

01























Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster














Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 3





21ndash40 Fand 16S





TI =sum119862

119894

ED50119894

(1)




3 Results





4 Archaea







1

724 plusmn 28

1

412 plusmn 220

1


1


1




1


1


1

370 plusmn 110


1


1


1

464 plusmn 231

1

1270 plusmn 984

1












119894



0

1

2

3

4

5

6

7

8

1 20 40 60 80 100 120 140

Num

ber O

TUs o

bser

ved


BuhCBuhDSliv



Archaea 5






01













Clus

ter A

Clus

ter B

I

II

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster





6 Archaea

01























Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster














Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Archaea







1

724 plusmn 28

1

412 plusmn 220

1


1


1




1


1


1

370 plusmn 110


1


1


1

464 plusmn 231

1

1270 plusmn 984

1












119894



0

1

2

3

4

5

6

7

8

1 20 40 60 80 100 120 140

Num

ber O

TUs o

bser

ved


BuhCBuhDSliv



Archaea 5






01













Clus

ter A

Clus

ter B

I

II

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster





6 Archaea

01























Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster














Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 5






01













Clus

ter A

Clus

ter B

I

II

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster





6 Archaea

01























Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster














Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Archaea

01























Clus

ter I

Clus

ter I

ICl

uste

r III

Gro

up 1

1b

Nitro

sosp

haer

a clu

ster














Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 7


4 Discussion










8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Archaea




5 Conclusions




Acknowledgment


References














Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Archaea 9






























10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

10 Archaea


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Phylogenetic Diversity of Archaea and the Archaeal Ammonia ...

Documents

Transcript of Phylogenetic Diversity of Archaea and the Archaeal Ammonia ...