Microarray and functional gene analyses of sulfate-reducing prokaryotes in low-sulfate, acidic fens...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2004, p. 6998–7009 Vol. 70, No. 120099-2240/04/$08.00�0 DOI: 10.1128/AEM.70.12.6998–7009.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Microarray and Functional Gene Analyses of Sulfate-ReducingProkaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence

of Recognized Genera and Novel LineagesAlexander Loy,1 Kirsten Kusel,2 Angelika Lehner,3 Harold L. Drake,2

and Michael Wagner1*Department of Microbial Ecology, Institute of Ecology and Conservation Biology, University of Vienna, Vienna,

Austria,1 and Department of Ecological Microbiology, University of Bayreuth, Bayreuth,2 andDepartment of Microbiology, Technical University of Munich, Freising,3 Germany

Received 9 March 2004/Accepted 27 July 2004

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountainsin Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facil-itate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (con-centration in the soil solution, 20 to 200 �M) and the acidic conditions (soil and soil solution pHs, approx-imately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schloppnerbrunnen I and II)of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide mi-croarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Mem-bers of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus De-sulfomonile were observed only in soils from Schloppnerbrunnen I. These results were confirmed and extendedby comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB)gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray anal-ysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groupsin the fens were formulated based on the known physiological properties of their cultured relatives. In addition tothese recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs weredetected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel,deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.

The dissimilatory reduction of sulfate is carried out exclu-sively by prokaryotic organisms and is one of the most impor-tant mineralization processes in anoxic aquatic environments,especially marine sediments (29, 30). In contrast to well-stud-ied sulfate-reducing communities in marine (18, 19, 38, 41, 53,56, 57, 72) and freshwater habitats (39, 40, 59, 60), relativelylittle is known about the distribution, diversity, and in situactivities of sulfate-reducing prokaryotes (SRPs) in terrestrialecosystems. The contribution of terrestrial SRPs to the overallturnover of organic matter is likely of minor importance on aglobal scale. However, SRPs contribute to the biodegradationof pollutants in soils and subsurface environments (1, 15, 49,71) and are important to the geomicrobiology of specializedterrestrial habitats that are subject to flooding, such as ricefields (68, 76, 77) and fens (3, 5).

�34S values and 35S-labeling patterns indicate that the dis-similatory reduction of sulfate is an ongoing process in theacidic fens of a forested catchment in northern Bavaria, Ger-many (Lehstenbach, Fichtelgebirge) (3, 5). The deposition ofsulfur that originated from the combustion of soft coal inEastern Europe (10) led to accumulation of sulfur in the soilsof this catchment (4). Although pollution controls have less-ened the deposition in recent years, desorption of sulfate in

aerated upland soils causes sulfate to enter fens at lower ele-vations. It was hypothesized that the dissimilatory reduction ofsulfate in these mainly anoxic, waterlogged acidic fen soils (thepH of the fen soils is approximately 4) contributes to the retentionof sulfur in this ecosystem (3, 4, 50). The reduction of sulfate inthese fens is also a sink for protons and thus decreases the acidityof the soil solution and groundwater of this habitat.

The acidity and low sulfate content of some of the fens in theLehstenbach catchment provide an unusual habitat for SRPs,and the occurrence and activity of these organisms in suchhabitats have received little attention. The main objectives ofthis study were (i) to assess the capacity of the fen soils toreduce sulfate along vertical soil profiles in the upper peatlayers, (ii) to determine the vertical community profiles forall known SRP lineages that inhabit the fens by the use ofa 16S rRNA-based oligonucleotide microarray (SRP-Phylo-Chip) (44), (iii) to resolve the possible existence of novel SRPlineages in the fens by retrieval of dsrAB, which are genes thatencode the alpha and beta subunits of the siroheme dissimila-tory (bi)sulfite reductase (EC 1.8.99.3) (34, 66, 74), and (iv) todeduce the possible in situ functional relationships that can beinferred from this collective information.

MATERIALS AND METHODS

Site description. The two low-moor fens, designated Schloppnerbrunnen I(50°08�14�N, 11°53�07�E) and Schloppnerbrunnen II (50°08�38�N, 11°51�41�E),that were investigated are in the Lehstenbach catchment in the Fichtelgebirgemountains in northeastern Bavaria (Germany). The catchment has an area of 4.2km2, and the highest elevation is 877 m above sea level. Ninety percent of the

* Corresponding author. Mailing address: Abteilung MikrobielleOkologie, Institut fur Okologie und Naturschutz, Universitat Wien,Althanstr. 14, A-1090 Vienna, Austria. Phone: 43 1 4277 54390. Fax: 431 4277 54389. E-mail: [email protected].

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area is covered with Norway spruce (Picea abies [L.] Karst.) of different ages.Upland soils in the catchment are not water saturated, have developed fromweathered granitic bedrock, and are predominantly cambisols and cambic pod-sols (according to the Food and Agriculture Organization system). Considerableparts of the catchment (approximately 30%) are covered by minerotrophic fensor intermittent seeps. The annual precipitation in the catchment is 900 to 1,160mm, and the average annual temperature is 5°C.

Schloppnerbrunnen I is covered with patches of Sphagnum moss and spruce,and the soil is a fibric histosol and is usually water saturated; in years withextremely hot summer months, the upper soil can become dry. Schloppnerbrun-nen II is permanently water saturated and completely overgrown by the grassMolinia caerula. The soil of Schloppnerbrunnen II has a larger amount of bio-available Fe3� than the soil of Schloppnerbrunnen I has. The soil pHs of Sch-loppnerbrunnen I and II were approximately 3.9 and 4.2, respectively; the soilsolution pH varied between 4 and 6.

Dialysis chambers. A soil solution from the upper 40 cm of each site wassampled with dialysis chambers (27) every 2 months from July 2001 to November2002. Each dialysis chamber consisted of 40 1-cm cells covered with a celluloseacetate membrane with a pore diameter of 0.2 �m. Prior to installation, thechamber was filled with anoxic, deionized water. The dialysis chambers wereplaced in the water-saturated fens for 2 weeks prior to sampling. On the samplingdate, each chamber was closed (i.e., made airtight), transported to the labora-tory, and sampled with argon-flushed syringes.

Collection of soil. For microcosms, soil samples from three different depths(approximately 0 to 10, 10 to 20, and 20 to 30 cm) were obtained in December2001 in sterile airtight vessels, transported to the laboratory, and processedwithin 4 h. For isolation of DNA, soil cores (diameter, 3 cm) from four differentdepths (approximately 0 to 7.5, 7.5 to 15, 15 to 22.5, and 22.5 to 30 cm) werecollected on 24 July 2001 and immediately cooled on ice. Soil samples werebrought to the laboratory, where they were diluted 1:1 (vol/vol) in phosphate-buffered saline (130 mM NaCl, 10 mM NaH2PO4, 10 mM Na2HPO4; pH 7.3),homogenized by vortexing, and stored at �20°C.

Anoxic microcosms. Thirty-gram (fresh weight) portions of soil were placedinto 125-ml infusion flasks (Merck ABS, Dietikon, Switzerland) inside an O2-freechamber (100% N2 gas phase), and 60-ml portions of anoxic, deionized waterwere added to facilitate sampling with sterile, argon-flushed syringes. The bottleswere closed with rubber stoppers and screw caps and were incubated in the darkat 15°C. Sulfate was added from a sterile anoxic stock solution (0.5 M K2SO4) toa final concentration of 500 �M. Microcosms were prepared in triplicate and hadinitial pHs that ranged from 5 to 5.5. The rates of sulfate consumption and CH4

formation were determined for 17 days by linear regression analysis of theconcentrations of sulfate and CH4, respectively. For CH4 formation rates, allconcentration data were included for linear regression analysis. For sulfate con-sumption rates, only the parts of the curves for when part of the supplementedsulfate was still available for consumption were used.

Analytical methods. pH was measured with a U457-S7/110 combination pHelectrode (Ingold, Steinbach, Germany). The sulfate content was determined byion chromatography (37). The concentration of CH4 in the headspace was mea-sured with a 5980 series II gas chromatograph (Hewlett-Packard Co., Palo Alto,Calif.) (37). Total reduced inorganic sulfur (TRIS) and acid volatile sulfur (AVS)

contents were determined by using previously described protocols (73). TRIS isassumed to be composed of pyrite (FeS2), amorphous FeS, and S0; AVS isamorphous FeS.

Extraction of DNA. DNA was extracted from soil homogenates by a modifi-cation (44) of a previously described protocol (25). The amount of extractedDNA was determined spectrophotometrically by measuring the absorbance at260 nm.

PCR amplification of 16S rRNA genes and dsrAB. PCR amplification of geneswas performed with 5 ng of environmental DNA. For subsequent microarrayhybridization, bacterial 16S rRNA gene fragments from soil genomic DNA wereamplified by using the primer pairs 616V-630R and 616V-1492R (Table 1), andPCR products were mixed prior to labeling. For confirmation of microarrayresults, 16S rRNA gene fragments of defined SRP groups were directly amplifiedfrom soil DNA by using previously described and newly designed primers (Table1). In addition, an approximately 1.9-kb dsrAB fragment was amplified from fensoil DNA by using the degenerate primers DSR1Fmix (equimolar mixture ofDSR1F, DSR1Fa, and DSR1Fb) and DSR4Rmix (equimolar mixture of DSR4R,DSR4Ra, DSR4Rb, and DSR4Rc) (Table 2).

Both positive controls (purified DNA from suitable reference organisms) andnegative controls (no DNA) were included in all PCR amplification experiments.For 16S rRNA gene and dsrAB amplifications, reaction mixtures (total volume,50 �l) containing each primer at a concentration of 25 pM were prepared byusing 10� Ex Taq reaction buffer and 2.5 U of Ex Taq polymerase (TakaraBiomedicals, Otsu, Shiga, Japan). Additionally, 20 mM tetramethylammoniumchloride (Sigma, Deisenhofen, Germany) was added to each amplification mix-ture to enhance the specificity of the PCR (35). Thermal cycling was carried outby using an initial denaturation step at 94°C for 1 min, followed by 30 (16S rRNAgenes) or 35 cycles (dsrAB) of denaturation at 94°C for 40 s, annealing attemperatures from 48 to 62°C (depending on the primer pair [Tables 1 and 2])for 40 s, and elongation at 72°C for 1.5 min. The cycling was completed by a finalelongation step at 72°C for 10 min. The presence and sizes of the amplificationproducts were determined by agarose (1%) gel electrophoresis. Ethidium bro-mide-stained bands were digitally recorded with a video documentation system(Cybertech, Hamburg, Germany).

DNA microarray analyses. Fluorescence labeling of PCR products, manufac-turing and processing of SRP-PhyloChips, reverse hybridization on microarrays,and scanning and image analyses of microarrays were performed as previouslydescribed (44). Spots that had signal-to-noise ratios equal to or greater than 2.0were considered positive. Oligonucleotides used for printing of the SRP-Phylo-Chips were obtained from MWG Biotech (Ebersberg, Germany). For each siteand soil depth, two separate microarrays with duplicate spots for each probewere hybridized with labeled PCR products. The sequences and specificities ofall probes are listed in the DNA microarray section of the probeBase website(43) http://www.microbial-ecology.net/probebase/.

Microarray hybridization patterns for the different depths of the samples fromthe two fens were used to infer binary similarities in order to provide a quanti-tative measure for comparison of hybridization data. The Jaccard coefficient (CJ)and the Sorenson coefficient (CS) for two samples were calculated by using thefollowing formulas: CJ � 100 � c � (a � b � c)�1 and CS � 100 � 2c � (a �b)�1, where a is the number of positive SRP-PhyloChip probes in the first

TABLE 1. 16S rRNA gene-targeted primers

Short namea Full nameb Annealingtemp (°C) Sequence (5�-3�) Specificity Reference

616V S-D-Bact-0008-a-S-18 52 AGA GTT TGA TYM TGG CTC Most Bacteria 32630R S-D-Bact-1529-a-A-17 52 CAK AAA GGA GGT GAT CC Most Bacteria 321492R S-�-Proka-1492-a-A-19 52, 60c GGY TAC CTT GTT ACG ACT T Most Bacteria and Archaea 33ARGLO36F S-G-Arglo-0036-a-S-17 52 CTA TCC GGC TGG GAC TA Archaeoglobus spp. 44DSBAC355F S-�-Dsb-0355-a-S-18 60 CAG TGA GGA ATT TTG CGC Most “Desulfobacterales” and

“Syntrophobacterales”61

DSMON85F S-G-Dsmon-0085-a-S-20 62 CGG GGT RTG GAG TAA AGT GG Desulfomonile spp. This studyDSMON1419R S-G-Dsmon-1419-a-A-20 62 CGA CTT CTG GTG CAG TCA RC Desulfomonile spp. This studySYBAC�282F S-�-Sybac-0282-a-S-18 60 ACG GGT AGC TGG TCT GAG “Syntrophobacteraceae” and

some other BacteriaThis study

SYBAC1427R S-�-Sybac-1427-a-A-18 60 GCC CAC GCA CTT CTG GTA “Syntrophobacteraceae” This studyDBACCA65F S-S-Dbacca-0065-a-S-18 58 TAC GAG AAA GCC CGG CTT Desulfobacca acetoxidans This studyDBACCA1430R S-S-Dbacca-1430-a-A-18 58 TTA GGC CAG CGA CAT CTG Desulfobacca acetoxidans This study

a Short name used in the reference or in this study.b Name of 16S rRNA gene-targeted oligonucleotide primer based on established nomenclature (6).c The annealing temperature was 52°C when the primer was used with forward primer 616V or ARGLO36F, and the annealing temperature was 60°C when the

primer was used with forward primer DSBAC355F.

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sample, b is the number of positive SRP-PhyloChip probes in the second sample,and c is the number of SRP-PhyloChip probes positive in both samples. Thesecoefficients are usually calculated based on presence-absence data for species inthe ecosystems compared. In this context, it should be noted that due to themultiple-probe design strategy of the SRP-PhyloChip, the number of positiveprobe signals on the microarray is generally much higher than the number ofspecies actually detected in the sample analyzed. Therefore, the calculated co-efficients can only be interpreted as indications of the similarity of hybridizationpatterns.

Cloning and sequencing. Prior to cloning, the PCR amplification productswere purified by low-melting-point agarose (1.5%) gel electrophoresis (NuSieve3:1; FMC Bioproducts, Biozym Diagnostics GmbH, Oldendorf, Germany) andstained in a SYBR Green I solution (10 �l of SYBR Green I [Biozym Diagnos-tics GmbH] in 100 ml of Tris-acetate-EDTA buffer [40 mM Tris, 10 mM sodiumacetate, 1 mM EDTA; pH 8.0]) for 45 min. Bands of the expected size wereexcised from the agarose gel with a glass capillary and melted with 80 �l ofdouble-distilled water for 10 min at 80°C. Four microliters of each solution wasligated as recommended by the manufacturer (Invitrogen Corp.) either into thecloning vector pCR2.1 of a TOPO TA cloning kit (16S rRNA gene PCR prod-ucts) or into the cloning vector pCR-XL-TOPO of a TOPO XL cloning kit(dsrAB PCR products). Nucleotide sequences were determined by a modificationof the dideoxynucleotide method (58) as described previously (54). In addition,internal dsrAB-targeted sequencing primers (Table 2) were used to complete thedsrAB sequences.

Phylogeny. Phylogenetic analyses were performed by using the alignment andtreeing tools implemented in the ARB program package (46). New 16S rRNAgene sequences were added to an ARB alignment of about 20,000 small-subunitrRNA gene sequences (which included sequences from all recognized SRPs andclone sequences from uncultured prokaryotes from sulfate-reducing environ-ments) by using the alignment tool ARB_EDIT. Alignments were refined byvisual inspection. 16S rRNA gene phylogenetic analyses were performed exclu-sively with sequences having more than 1,150 bases by using distance matrix,maximum-parsimony, and maximum-likelihood methods (45). The compositionof the 16S rRNA gene sequence data sets varied with respect to the referencesequences and alignment positions. The variability of the individual alignmentpositions was determined by using the ARB_SAI tools and was used as a crite-rion to remove or include variable positions for phylogenetic analyses.

New dsrAB sequences were added to an ARB alignment that contained alldsrAB sequences of recognized SRPs (22, 34, 74) and uncultured SRPs availablein the GenBank database (9). Deduced amino acid sequences were manuallyaligned by using the editor GDE 2.2 (64). Nucleic acid sequences were alignedaccording to the alignment of amino acids. For phylogenetic inference of DsrABamino acid sequences, insertions and deletions were removed from the data setby using a suitable alignment mask (indel filter), which left a total of 543 amino

acid positions (alpha subunit, 327 positions; beta subunit, 216 positions) forcomparative analyses. Distance matrix (using FITCH with global rearrangementsand randomized input order of species) and maximum-parsimony trees werecalculated with the Phylogeny Inference Package (PHYLIP) (21). In addition,the programs MOLPHY (2) and TREE-PUZZLE (67) were used to infer max-imum-likelihood trees with JTT-f as the amino acid replacement model. Todetermine the level of amino acid identity between two DsrAB sequences,ambiguous amino acid positions and the alignment regions of insertions anddeletions (indel filter) were omitted.

Parsimony bootstrap analyses for nucleic acid (16S rRNA gene) and protein(DsrAB) trees were performed with PHYLIP. One hundred bootstrap resam-plings were analyzed for each calculation. All phylogenetic consensus trees weredrawn by using established protocols (45).

Bacterial nomenclature. The names of bacterial taxa used here are in accor-dance with the prokaryotic nomenclature proposed in the taxonomic outline ofBergey’s Manual of Systematic Bacteriology, 2nd ed. (23; http://dx.doi.org/10.1007/bergeysoutline200210).

Nucleotide sequence accession numbers. The sequences determined in thisstudy have been deposited in the GenBank database under accession numbersAY167444 to AY167462 (16S rRNA gene clones) and AY167464 to AY167483(dsrAB clones).

RESULTS

Oxidized and reduced inorganic sulfur in fen soils. Theconcentrations of sulfate in the soil solutions from Schloppner-brunnen I and Schloppnerbrunnen II varied over the year; theminimum concentration was 20 �M in late autumn, and themaximum concentration was 200 �M after the snow melt inFebruary. At Schloppnerbrunnen I, the average concentrationsof TRIS in triplicate soil samples obtained from depths of 0 to10, 10 to 20, and 20 to 30 cm in December were approximately0.05 �mol g (fresh weight) of soil�1 at each depth. In contrast,the average concentration of AVS increased from 0.01 to 0.05�mol g (fresh weight) of soil�1 as the soil depth increased. AtSchloppnerbrunnen II, the average concentrations of TRISwere approximately 0.29, 0.47, and 0.63 �mol g (fresh weight)of soil�1 at depths of 0 to 10, 10 to 20, and 20 to 30 cm,respectively; in contrast, the average concentrations of AVSwere more uniform and were approximately 0.05, 0.06, and

TABLE 2. Dissimilatory (bi)sulfite reductase gene (dsrAB)-targeted primersa

Primer Sequence (5�-3�) Specificity Reference

DSR1Fb ACS CAC TGG AAG CAC G Archaeoglobus fulgidus, Archaeoglobus profundus, Desulfovibrio vulgaris 74DSR1Fab ACC CAY TGG AAA CAC G Desulfotomaculum thermocisternum, Desulfobulbus rhabdoformis,

Desulfobacter vibrioformisThis study

DSR1Fbb GGC CAC TGG AAG CAC G Thermodesulforhabdus norvegica This studyDSR4Rb GTG TAG CAG TTA CCG CA Archaeoglobus fulgidus, Desulfovibrio vulgaris, Desulfobulbus rhabdoformis 74DSR4Rab GTG TAA CAG TTT CCA CA Archaeoglobus profundus This studyDSR4Rbb GTG TAA CAG TTA CCG CA Desulfobacter vibrioformis This studyDSR4Rcb GTG TAG CAG TTK CCG CA Thermodesulforhabdus norvegica, Desulfotomaculum thermocisternum This studyDSR978Fac GGT CAT CGA CCT TTG TCC Schloppnerbrunnen I soil OTU 5 This studyDSR978Fbc CGT CGT CGG GAA GTG CCC Schloppnerbrunnen I soil OTU 8 This studyDSR978Fcc AGT AGT CGA CCT TTG CCC Schloppnerbrunnen I and II soil OTU 6 This studyDSR978Fdc TGT CAC CGA TCT CTG CCC Schloppnerbrunnen I soil OTU 1 This studyDSR978Fec TGT TAC CGA CCT CTG CCC Schloppnerbrunnen II soil OTU 1 (dsrSbII-20) This studyDSR978Ffc TGT CAC CGA TCT TTG CCC Schloppnerbrunnen II soil OTU 4 (dsrSbII-15) This studyDSR978Fgc CGT CAC CAT TCT CTG CCC Schloppnerbrunnen II soil OTU 4 (dsrSbII-9) This studyDSR978Fhc GGT CGT TGA CAT GTG TCC Schloppnerbrunnen II soil OTU 11 This studyDSR978Fic GGT CTG CAA TCT CTG YCC Schloppnerbrunnen I and II soil OTU 2 and 3 This studyDSR978Fjc GGT TGT TGA CCT TTG CCC Schloppnerbrunnen I soil OTU 9 This studyDSR978Fkc CGT TTG CGA TCT CTG CCC Schloppnerbrunnen II soil OTU 7 This studyDSR860Fc AGA TCC GGC GGG ACG ATG Schloppnerbrunnen I soil OTU 10 This study

a The target sites of all DSR1 and DSR4 primers were analyzed for the SRPs (n � 7) for which complete dsrAB operons were available in the GenBank database(9). SRPs with a target sites fully complementary to the primers are indicated.

b The primer was used under nonstringent conditions by using an annealing temperature of 48°C for PCR in order to target a wide diversity of SRPs.c Internal sequencing primer used to complete dsrAB sequences retrieved from acidic fen sites at Schloppnerbrunnen I and II.

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0.05 �mol g (fresh weight) of soil�1 at the three depths, re-spectively.

Capacity of fen soils to consume sulfate. Supplemental sul-fate was rapidly consumed without an apparent delay in anoxicmicrocosms that contained soil from the upper peat layers(Fig. 1). Soils from the three depths yielded similar concentra-tions of TRIS and AVS (Table 3). The average concentrationsof TRIS and AVS were 0.67 and 0.12 �mol g (fresh weight) ofsoil�1, respectively, in sulfate-supplemented Schloppnerbrun-

nen I microcosms at the end of incubation. The average con-centrations of TRIS and AVS in unsupplemented controlswere 0.16 and 0.03 �mol g (fresh weight) of soil�1, respec-tively. The Schloppnerbrunnen II soils yielded results similarto those obtained with the Schloppnerbrunnen I soils, althoughthe values were slightly higher. However, the average amountof reduced sulfur recovered was only approximately 18% ofthe amount of sulfate-derived sulfur that was consumed. Partof the reduced sulfur might have been lost in the headspaceas H2S due to the low soil pH. Despite this discrepancy, theconcentrations of TRIS and AVS in soils were higher at theend of incubation in sulfate-supplemented microcosms than atthe end of incubation in unsupplemented controls, indicatingthat the consumption of supplemental sulfate was linked to thedissimilatory reduction of sulfate.

The maximum rates at which supplemental sulfate was con-sumed in the soil microcosms prepared from soils obtained atdepths of 0 to 10, 10 to 20, and 20 to 30 cm from Schloppner-brunnen I were approximately 0.14, 0.11, and 0.14 �mol g(fresh weight) of soil�1 day�1, respectively, and the corre-sponding rates for soils from Schloppnerbrunnen II were 0.41,0.13, and 0.13 �mol g (fresh weight) of soil�1 day�1, respec-tively. In the sulfate-supplemented Schloppnerbrunnen I soilmicrocosms the rates of CH4 production were 0.02, 0.01, and0.01 �mol g (fresh weight) of soil�1 day�1, respectively, andthe rates in the Schloppnerbrunnen II soil microcosms were 0.06,0.03, and 0.02 �mol g (fresh weight) of soil�1 day�1, respectively;the rates of production of CH4 in control Schloppnerbrunnen Imicrocosms not supplemented with sulfate were 0.07, 0.04, and0.04 �mol g (fresh weight) of soil�1 day�1, respectively, andthe rates in the corresponding Schloppnerbrunnen II micro-cosms were 0.19, 0.09, and 0.09 �mol g (fresh weight) of soil�1

day�1, respectively (Fig. 2 and data not shown). Thus, supple-mental sulfate caused a 71% decrease in the average rate atwhich CH4 was produced.

16S rRNA-based phylogenetic profiles of SRPs at differentfen soil depths. All soil depths at Schloppnerbrunnen I yieldedsimilar microarray hybridization patterns (Fig. 3A), indicatingthat there were minimal depth-dependent changes in the rich-ness of recognized SRP phylotypes. Consistent with this obser-vation, high Jaccard and Sorensen coefficient values (56 to 83%and 71 to 91%, respectively) were inferred by comparing thehybridization patterns for the different soil depths (Table 4).The microarray results indicated the presence of (i) Desulfo-monile spp., (ii) some species of the family “Desulfobacteraceae,”

FIG. 1. Consumption of supplemental sulfate (500 �M) in anoxicmicrocosms of soil obtained from Schloppnerbrunnen I (A) and II (B).The values are averages standard deviations for triplicate determi-nations.

TABLE 3. Effect of the consumption of supplemental sulfate on the formation of reduced sulfur compounds inanoxic microcosms of soil obtained from Schloppnerbrunnen I and IIa

Sampling site Depth(cm)

Concn (�mol g [fresh wt]�1)

AVSb AVS control (no sulfate added) TRISc TRIS control (no sulfate added)

Schloppnerbrunnen I 0–10 0.057 0.023 0.031 0.004 0.648 0.493 0.101 0.02010–20 0.127 0.051 0.023 0.003 0.646 0.192 0.146 0.16820–30 0.167 0.100 0.040 0.024 0.716 0.389 0.234 0.051

Schloppnerbrunnen II 0–10 0.389 0.011 0.046 0.006 0.586 0.158 0.174 0.03710–20 0.147 0.020 0.058 0.017 0.786 0.066 0.477 0.16420–30 0.295 0.089 0.052 0.015 1.202 0.353 0.530 0.234

a The data are averages standard deviations for triplicate soil samples obtained at the end of incubation.b AVS is amorphous FeS.c TRIS is amorphous FeS, S0, and FeS2.



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and (iii) bacteria belonging to the Syntrophobacter-Desulfo-virga-Desulforhabdus line of descent of the family “Syntropho-bacteraceae” (order “Syntrophobacterales”) (Fig. 3B). Archaeo-globus 16S rRNA genes were not detected by PCR with genus-specific primers (Table 1). Consistent with the microarraypatterns, direct PCR of Schloppnerbrunnen I soil DNA withDesulfomonile- and “Syntrophobacteraceae”-specific primerpairs (Table 1) yielded increasing amounts of PCR products ofthe expected size with increasing soil depth (data not shown).Cloning and sequencing of the PCR products from soil ata depth of 22.5 to 30 cm confirmed that Desulfomonile spp.and Syntrophobacter wolinii-related bacteria were present atSchloppnerbrunnen I (Fig. 4).

The microarray hybridization patterns for soils from differ-ent depths at Schloppnerbrunnen II did not vary a great deal(Fig. 5A and Table 4), but they were less complex than thoseof the Schloppnerbrunnen I soils. The binary similarities of thehybridization patterns obtained for the different depths yieldedhigh Jaccard and Sorensen coefficient values, which were com-parable to the values obtained for the samples from differentdepths at Schloppnerbrunnen I (Table 4). Only probes target-ing SRPs at higher taxonomic levels (e.g., DELTA495a [micro-array positions C2 and E2] and DSBAC355 [microarray posi-tion C7]) were unambiguously positive. However, the mean

FIG. 2. Effect of the consumption of supplemental sulfate (500�M) on the production of methane in anoxic microcosms of soil ob-tained from Schloppnerbrunnen I. The values are averages standarddeviations for triplicate determinations. Open symbols, methane pro-duction with supplemental sulfate; solid symbols, controls (no sulfateadded); circles, 0 to 10 cm, triangles, 10 to 20 cm; squares, 20 to 30 cm.

FIG. 3. (A) Use of SRP-PhyloChip for surveys of SRP diversity at four different depths at Schloppnerbrunnen I. Each probe was spotted induplicate. The specificity and microarray position of each probe have been described previously (44). Probe spots having a signal-to-noise ratiosequal to or greater than 2.0 are indicated by boldface boxes and were considered to be positive. The dotted boldface boxes indicate that only oneof the duplicate spots had a signal-to-noise ratio equal to or greater than 2.0. (B) Flow chart illustrating the presence of distinct SRP groups inSchloppnerbrunnen I as inferred from positive signals for sets of probes with nested and/or parallel specificity. For each probe the position on themicroarray is indicated by a superscript.



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signal-to-noise ratios of the probe spots that were specific formembers of the Syntrophobacter-Desulfovirga-Desulforhabduslineage were just below the threshold value (Fig. 5B). DirectPCR of Schloppnerbrunnen II soil DNA with “Syntrophobac-teraceae”-specific primer pairs (Table 1) yielded increasingamounts of PCR products of the expected size with increasingsoil depth (data not shown) and verified the microarray patterns(data not shown). Cloning and sequencing of the PCR prod-ucts from soil obtained at a depth of 22.5 to 30 cm confirmedthat S. wolinii-related bacteria were present at Schloppner-brunnen II (Fig. 4).

The primer pair DSBAC355F-1492R is specific for mostmembers of the orders “Desulfobacterales” and “Syntrophobac-terales” (Table 1). This primer pair was used to screen for SRPsbelonging to these orders which were not covered by thespecific primer pairs described above. Twelve DSBAC355F-1492R-dependent clone sequences were determined for thedeepest soil samples from each field site, but none of thesesequences were closely related to recognized SRP 16S rRNAgene sequences (data not shown).

dsrAB diversity in fen soils. As observed for SRP 16S rRNAgene PCR products (see above), the amount of dsrAB PCRproducts increased as the soil depth increased (data notshown). The dsrAB PCR products that were retrieved from thedeepest soils (22.5 to 30 cm) from Schloppnerbrunnen I and IIwere used to construct dsrAB clone libraries; the clone librariesfor Schloppnerbrunnen I and II were designated dsrSbI anddsrSbII, respectively; 41 of 42 randomly picked dsrSbI clonesand 35 of 48 randomly picked dsrSbII clones had an insert ofthe expected size (1.9 kb). However, partial sequencing fol-lowed by BLAST analyses (7) demonstrated that only 29 clonesfrom the dsrSbI library and 24 clones from the dsrSbII librarycontained dsrAB sequences. Comparative sequence analysis ofthe partial DsrAB sequences grouped the 53 Schloppnerbrun-nen dsrAB clones in 11 clusters. The complete sequence of atleast one dsrAB clone per cluster was subsequently deter-mined. All dsrAB clones with deduced DsrAB amino acid se-quence identities equal to or greater than 90% with each otherwere grouped into an operational taxonomic unit (OTU). Thisgrouping yielded 11 OTUs for both libraries (Table 5). ThreeOTUs (OTUs 1, 3, and 6) were present at both fen sites; incontrast, eight OTUs contained dsrAB clones that were foundonly at either Schloppnerbrunnen I or Schloppnerbrunnen II(four OTUs each).

The phylogenetic affiliations of deduced DsrAB sequencesare shown in Fig. 6. OTU 1, which comprised most of thedsrSbI clones, and the dsrSbII-specific OTU 4 displayed highsequence identity to the OTU of a groundwater clone derivedfrom a uranium mill tailings site (Table 5). Clones in OTUs 1and 4 formed a stable, monophyletic group with the deltapro-teobacterial SRP Desulfobacca acetoxidans. One dsrAB clonederived from each fen site formed OTU 6, which was closelyrelated to S. wolinii. OTU 9, which consisted of a single clonederived from Schloppnerbrunnen I and was affiliated with De-sulfomonile tiedjei, constituted an additional deltaproteobacte-rial lineage. OTU 5, which was composed of two clones derivedfrom Schloppnerbrunnen I, formed an independent branchwithin a monophyletic deltaproteobacterial cluster consistingof the family “Desulfobacteraceae” and different groups belong-ing to the order “Desulfovibrionales.” The remaining six OTUsformed three deeply branching evolutionary lines of descentthat were different from any cultured SRP lineage (Fig. 6).One of these three deeply branching lines of descent con-tained OTUs 2, 7, 8, and 10; the other two lines of descentcontained either OTU 3 or OTU 11. OTUs 2 and 7 consistedexclusively of dsrSbII clones. In contrast, only dsrSbI cloneswere present in OTUs 8 and 10. Interestingly, each of thesethree deeply branching lines of descent also contained at leastone dsrAB clone that was derived from uranium mill tailingsgroundwater (16). It is also noteworthy that a dsrAB clonerelated to OTU 10 was recently retrieved from Everglades soil(14).

Molecular assessment of D. acetoxidans-related SRPs. TheDsrAB analyses indicated that fen soils at both Schloppner-brunnen I and II harbored D. acetoxidans-related SRPs.Because no species-specific probe for D. acetoxidans wasincluded on the microarray, a 16S rRNA-based PCR assaywith the D. acetoxidans-specific primer pair DBACCA65F-DBACCA1430R (Table 1) was used to retrieve 16S rRNAgene sequences of D. acetoxidans-related bacteria. PCRs werecarried out at low stringency to allow amplification of 16SrRNA genes of D. acetoxidans-related SRPs even if they hadmismatches in the primer target sites. Although this primerpair produced a PCR product of the expected size (1.4-kb)when D. acetoxidans was used as a positive control, it did notyield any D. acetoxidans-specific PCR product from Schlopp-nerbrunnen I or II soils (data not shown).

TABLE 4. Similarity matrix for SRP communities in the fen samples based on the presence or absence of SRP-PhyloChip probe signalsfor soil samples taken from four depths at Schloppnerbrunnen I and II

Sample Jaccard coefficient (%)/Sorenson coefficient (%)

Sitea Depth SbI, 0–7.5 cm SbI, 7.5–15 cm SbI, 15–22.5 cm SbI, 22.5–30 cm SbII, 0–7.5 cm SbII, 7.5–15 cm SbII, 22.5–30 cm

SbI 0–7.57.5–15 83/9115–22.5 59/74 56/71

22.5–30 65/79 62/76 68/81

SbII 0–7.5 53/69 56/72 35/51 35/517.5–15 56/71 59/74 37/54 37/54 67/8015–22.5 53/69 56/72 35/51 35/51 80/89 82/90

22.5–30 41/58 44/61 27/42 27/42 78/88 64/78 78/88

a SbI, Schloppnerbrunnen I; SbII, Schloppnerbrunnen II.



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DISCUSSION

SRP activities in fen soils. The occurrence of TRIS and AVSin the upper peat layers and the capacity of the soils to con-sume sulfate and form TRIS and AVS under anoxic conditionscorroborated previous results (3, 5) that indicated that thedissimilatory reduction of sulfate is an on-going process in the

low-sulfate, acidic fens of the Lehstenbach catchment. How-ever, the maximum concentrations of sulfate detected in thesoil solution never exceeded 200 �M, and the low concentra-tions (20 �M) of sulfate in the soil solution obtained in autumnmight be insufficient for the dissimilatory reduction of sulfate(42). It can be projected that the low concentration of sulfate

FIG. 4. 16S rRNA gene phylogenetic consensus tree based on neighbor-joining analysis performed with a 50% conservation filter for theDeltaproteobacteria. The tree shows the affiliations of clone sequences from Schloppnerbrunnen I and II soils (indicated by boldface type). Bar �10% estimated sequence divergence. Polytomic nodes connect branches for which a relative order could not be determined unambiguously byapplying distance matrix, maximum-parsimony, and maximum-likelihood treeing methods. Parsimony bootstrap values for branches are indicatedby solid circles (90%) and open circles (75 to 90%). Branches without circles had bootstrap values of less than 75%. Brackets indicate theperfect-match target organisms of the probes. The microarray position is indicated by a superscript after each probe designation. Cadagno Lakeclones were not sequenced at the target site for probe DSMON1421. TCB, trichlorobenzene.



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is a limiting seasonal factor for the sulfate-reducing activity ofSRPs in these fens.

Fens of the Lehstenbach catchment emit 0.02 to 15 mmol ofCH4 m�2 day�1 (28). Methanogenesis was inhibited in anoxicmicrocosms supplemented with sulfate; this result supportsprevious findings suggesting that SRPs outcompete methano-gens in peatlands under certain in situ conditions (11, 79). Therates of consumption of supplemental sulfate in soil micro-cosms were twice as high as the rates of formation of methanein microcosms lacking sulfate and were comparable to the ratesfor eutrophic waterlogged Everglades soils (14). These collec-tive observations indicate that sulfate, when available, is sub-ject to rapid reduction in these fens.

SRP diversity in fen soils. Both the 16S rRNA-based (mi-croarray and clone library) and dsrAB-based analyses indicatedthat the SRP community compositions of the two fen soils werenot identical. Soil from Schloppnerbrunnen I contained mem-bers of the genus Desulfomonile and the family “Syntropho-bacteraceae,” while only the latter group was detected in soilfrom Schloppnerbrunnen II. Only 7 and 4% of the dsrABclones from Schloppnerbrunnen I and II, respectively, wereaffiliated with these organisms. A large fraction of the remain-ing dsrAB clones (72 and 21% of the clones derived fromSchloppnerbrunnen I and II, respectively) formed a monophy-letic group with D. acetoxidans, a result that is paradoxical in

that this organism was not detected by nonstringent 16S rRNAgene amplification with D. acetoxidans-specific primers. Thefailure to detect the D. acetoxidans-like organisms via their 16SrRNA genes possibly reflects their moderate phylogeneticrelatedness to D. acetoxidans (73 to 75% DsrAB amino acididentity). Consequently, the D. acetoxidans-like organisms ofthe fens might have 16S rRNA genes that were not targetedby the D. acetoxidans-specific primers. An encompassing 16SrRNA gene library from the Schloppnerbrunnen sites obtainedwith general bacterial primers could help determine whethersuch organisms actually occur in the acidic fens. Alternatively,the D. acetoxidans-related dsrAB sequences might originatefrom SRPs which are phylogenetically not closely related toD. acetoxidans but received their dsrAB genes via lateral genetransfer from a D. acetoxidans-related donor. The fact that sev-eral horizontal dsrAB gene transfer events have been described(34; V. Zverlov, unpublished data) lends weight to this hypoth-esis.

The molecular analyses indicated that S. wolinii-related bac-teria are present in these acidic fen soils, a result that is con-sistent with detection of this species in an acid-tolerant metha-nogenic consortium derived from a Sphagnum peat bog (63).16S rRNA gene sequences obtained from rice paddy soil indi-cate that S. wolinii-like bacteria also occur in other types ofsoils (61). S. wolinii was first isolated from an anaerobic mu-

FIG. 5. (A) Use of SRP-PhyloChip for surveys of SRP diversity at four different depths at Schloppnerbrunnen II. See the legend to Fig. 3 foradditional details. (B) Flow chart illustrating the presence of distinct SRP groups in Schloppnerbrunnen II soil as inferred from positive signalsfor sets of probes with nested specificity. For each probe the position on the microarray is indicated by a superscript. The asterisk indicates thatthe mean signal-to-noise ratios of the duplicate SYBAC986 spots for 7.5 to 15, 15 to 22.5, and 22.5 to 30 cm were just below the threshold valueof 2.0 (1.88, 1.95, and 1.70, respectively).



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nicipal sewage digestor in syntrophic cocultures with methano-gens or SRPs (12) and oxidizes propionate to acetate and CO2.The electrons that are obtained from the oxidation of propi-onate are transferred via hydrogen and/or formate to meth-anogens that reduce CO2 to methane. The oxidation of pro-pionate is exergonic only when hydrogen and/or formate iscontinuously removed by the syntrophic, methanogenic part-ner. The discovery that S. wolinii is capable of dissimilatingsulfate led to its isolation (75) and proved that it was not anobligate syntroph (12). Due to the low concentration of sulfatein the Schloppnerbrunnen fens and the likelihood that H2 is animportant substrate for moderately acid-tolerant methanogensin this ecosystem (28), it is tempting to speculate that theS. wolinii-like bacteria of these fens also have a syntrophic life-style and interact with methanogens. However, the S. wolinii-related 16S rRNA gene sequences that were retrieved from thefen soils were 3.6 to 5.5% dissimilar to the 16S rRNA genesequence of S. wolinii; likewise, the levels of sequence dissim-ilarity among the S. wolinii-like fen clones ranged from 0.1 to5.2%. Thus, the cloned sequences represent several novelgenomospecies (65) that might have physiologies that are atleast partially dissimilar to that of S. wolinii.

Desulfomonile-related organisms appear to be members ofthe soil community at Schloppnerbrunnen I. Members of thisgenus occur in other low-sulfate systems, such as an alpine lake(8), a forested wetland (13), deep crystalline bedrock (52), anda uranium mill tailings site (16), but they also occur in a hy-persaline, sulfide-rich microbial mat (44). An environmentallyimportant feature of the two cultured species of this genus,Desulfomonile limimaris and Desulfomonile tiedjei, is their abil-ity to reductively dehalogenate anthropogenic compounds,such as polychlorinated biphenyls, perchloroethene, and chlo-

robenzenes (47, 48, 69). These species can also use H2 as anelectron donor (55, 69). Thus, Desulfomonile-related organ-isms might compete for H2 and limit hydrogenotrophic metha-nogenesis when sulfate becomes available in the Schloppner-brunnen I fen. However, the 16S rRNA gene sequences ofDesulfomonile-related clones were 5.2 to 7.5% dissimilar tothose of the cultured members of this genus, suggesting thatthe ecophysiological capacities of the organisms representedby the cloned sequences might not be identical to those ofknown Desulfomonile species.

D. acetoxidans-like organisms appear to be present in bothfens and have previously been detected in other low-sulfatehabitats (13, 14, 16). The only cultured member of the genusDesulfobacca, D. acetoxidans, was isolated from a laboratory-scale upflow anaerobic sludge bed reactor fed with acetate andsulfate. This organism is specialized in acetate consumptionand can outcompete acetoclastic methanogens (51). Becausethe D. acetoxidans dsrAB clone group was numerically thelargest clone group obtained, we hypothesized that D. acetoxi-dans-like bacteria are abundant in these fens and are at leastpartially responsible for the lack of acetate accumulation andlow abundance of acetoclastic methanogens in the fen soils (28).

SRP novelties in fen soils. In addition to molecular signa-tures of well-known SRP lineages, novel types of dissimilatory(bi)sulfite reductases were detected in both fens. The deducedDsrAB amino acid sequences were remarkably dissimilar tothose of cultured SRPs (the maximum deduced level of DsrABamino acid sequence identity between one of the clones[dsrSbII-36] and a cultured SRP [Desulfotomaculum alkaliphi-lum DSM 12247] was 68%) and formed six deeply branchingOTUs in the DsrAB tree (Fig. 6). Only one of these OTUs wasfound in both fens. The estimated diversity coverage of the

TABLE 5. OTUs of sulfate-reducing prokaryotes based on comparative sequence analyses of dsrAB retrieved fromacidic fen soil at the Schloppnerbrunnen I and II sampling sites

OTUaNo. of clonesb

dsrAB clonescMost similar dsrAB sequence in GenBank

as determined by BLAST search(accession no./% amino acid identity)

Inferred phylogenyd

dsrSbI dsrSbII

1 21 1 dsrSbI-56, -57, -58, -59, -60, 61-, -62,-65, -66, -67, -69, -72, -73, -74, -78,-79, -81, -83, -84, -86, and -87;dsrSbII-20

Uranium mill tailings clone UMTRAdsr828-17(AY015508, AY015597/86–89)

Desulfobacca acetoxidans related,Deltaproteobacteria

2 9 dsrSbII-3, -18, -21, -22, -23, -28, -34,-42, and -47

Everglades clone FISU-12 (AY096051/83-84) Unaffiliated with known SRPs

3 1 6 dsrSbI-71; dsrSbII-4, -5, -8, -12, -25,and -36

Uranium mill tailings clone UMTRA826-5(AY015548, AY015614/88)

Unaffiliated with known SRPs

4 4 dsrSbII-9, -11, -15, and -33 Uranium mill tailings clone UMTRAdsr828-17(AY015508, AY015597/87-89)

Desulfobacca acetoxidans related,Deltaproteobacteria

5 2 dsrSbI-82 and -50 “Desulfobacterium oleovorans” (AF418201/80) Deltaproteobacteria6 1 1 dsrSbI-54; dsrSbII-40 Syntrophobacter wolinii (AF418192/87-88) Syntrophobacter wolinii related,

Deltaproteobacteria7 2 dsrSbII-2 and -16 Everglades clone F1SU-12 (AY096051/83) Unaffiliated with known SRPs8 2 dsrSbI-75 and -85 Only distantly related sequences in GenBank Unaffiliated with known SRPs9 1 dsrSbI-88 Desulfomonile tiedjei (AF334595/85) Desulfomonile-related, Deltapro-

teobacteria10 1 dsrSbI-64 Uranium mill tailings clone UMTRAdsr626-20

(AY015569, AY015611/90)Unaffiliated with known SRPs

11 1 dsrSbII-39 Uranium mill tailings clone UMTRAdsr624-8(AY015519, AY015596/92)

Unaffiliated with known SRPs

a dsrAB clones exhibiting deduced DsrAB sequence identity equal to or greater than 90% were grouped in an OTU. The OTUs are listed and sequentially numberedaccording to the total number of clones retrieved.

b The dsrAB clone libraries for Schloppnerbrunnen I and II were designated dsrSbI and dsrSbII, respectively. Homologous coverage (C) was calculated as follows:C � [1 � (n1 � N�1)]� 100%, where n1 is the number of OTUs containing only one sequence and N is the total number of dsrAB clones analyzed (24, 31, 62). Thehomologous coverage for the 29 clones in dsrSbI was 86%, and the homologous coverage for the 24 clones in dsrSbII was 88%.

c Completely sequenced dsrAB clones (1,750 bases) are indicated by boldface type.d Phylogeny of dsrAB clones as inferred from Fig. 6.



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established dsrAB libraries was almost 90% (Table 5), whichsuggests that the actual diversity of dsrAB sequences (detect-able by the PCR approach used) present in the fens was largelyrecovered. Thus, the fact that five of the six deeply branchingdsrAB OTUs were not detected at both fen sites is indicative ofsite-specific occurrence of the organisms. On the other hand,three of the retrieved lineages displayed affiliations with dsrABsequences that were derived from a uranium mill tailings soil(16) or an Everglades soil (14), indicating that some of the de-tected lineages are widely distributed in low-sulfate ecosystems.

The novel types of dissimilatory (bi)sulfite reductases de-tected in the present study originated from SRPs that belong toeither previously undescribed phylogenetic groups or phyla notyet known to contain organisms capable of dissimilating sulfate

or sulfite. Certain type II methanotrophs harbor two differentgenes for subunit A of particulate methane monooxygenase(20, 70), and the novel dsrAB types could likewise representadditional dissimilatory (bi)sulfite reductases in well-knownSRPs. However, SRPs that possess multiple dsr operons withsignificantly different sequences have not been reported. The-oretically, the novel dsrAB types from the Schloppnerbrunnenfens could be pseudogenes whose sequences differ from rec-ognized dsrAB sequences due to a higher mutation frequencycaused by lack of selective pressure. However, there are threelines of evidence that argue against this possibility. (i) ThedsrAB sequences did not contain any unexpected stop codons.(ii) Both subunits of the deduced DsrAB amino acid sequencescontained the Cys motif consensus sequences Cys-X5-Cys and

FIG. 6. Phylogenetic consensus tree (based on FITCH analysis) for DsrAB amino acid sequences deduced from dsrAB sequences longer than1,750 bases, showing the affiliation of OTUs from Schloppnerbrunnen fen soils (indicated by boldface type). DsrAB sequences deduced from dsrABsequences shorter than 1,750 bases (indicated by dashed branches) were individually added to the distance matrix tree without changing the overalltree topology by using the ARB treeing tool PARSIMONY_INTERAKTIV. Bar � 10% estimated sequence divergence. See the legend to Fig.4 for additional details.



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Cys-X3-Cys that are essential for binding the [Fe4S4]-sirohemecofactor of (bi)sulfite reductase (17); as in other (bi)sulfitereductases (26, 34), the Cys-X5-Cys motif of some of the de-duced DsrAB amino acid sequences (OTUs 2, 7, 10, and 11)was truncated in the beta subunit. (iii) The nonsynonymous/synonymous substitution rate ratios of branches leading to thenew dsrAB sequence clusters are low (�0.1), suggesting thatthere was strong purifying (negative) selection indicative ofexpressed and functionally active proteins (78).

Conclusions In this study we assessed the potential activitiesand molecular signatures of SRPs in two acidic fens within theLehstenbach catchment. Although some of the geochemicalfeatures (e.g., pH, temperature, and low concentration of sul-fate) of these fens are similar, the types of vegetation in the twofens are different. In addition, only Schloppnerbrunnen II isenriched both with dithionite-extractable pedogenic iron ox-ides and oxalate-extractable poorly crystallized iron oxides(36). Poorly crystallized iron oxides are the favored reducibleforms of Fe3� for microbial reduction, and Fe3� might be analternative electron acceptor for the anaerobic oxidation oforganic matter at Schloppnerbrunnen II. The collective dataobtained in this study revealed stable diversity profiles forSRPs in the upper peat layers of the fen soils but also revealedsite-specific novel SRP lineages. Thus, one might speculatethat the different types of vegetation and different bioavail-abilities of iron of the two fens are important factors in deter-mining which SRPs become established in either one or bothfens. Likewise, the seasonal variability in the water content ofthe two fens might be a contributing factor. Resolving thegenomic and ecophysiological characteristics of the SRP com-munity members of these fens is a major challenge for futureresearch and could yield new insights into the novel physiolo-gies and structure-function relationships of resident SRPs thatenable them to compete in low-sulfate, acidic habitats.

ACKNOWLEDGMENTS

We thank Helga Gaenge, Sibylle Schadhauser, Silvia Weber, SandraPraßl, and Sonja Trenz for their excellent technical assistance. MichaelFriedrich is acknowledged for providing the dsrAB sequence of D.acetoxidans DSM 11109T (GenBank accession number AY167463).We also thank Matthias Horn for critical reading of the manuscriptand for helpful discussions and Michael Klein for help with dsrABprimer design. The tedious work of Michael Klein and Natuschka Leerequired for establishing and maintaining the dsrAB database is highlyappreciated.

This research was supported by grants from the bmb�f (01 LC 0021subproject 2 in the framework of the BIOLOG program to M.W. andPT BEO 51-0339476 D) and the Bayerische Forschungsstiftung (De-velopment of Oligonucleotide DNA Chips in cooperation with MWGBiotech; project 368/99 to M.W.).

REFERENCES

1. Abdelouas, A., W. Lutze, W. Gong, E. H. Nuttall, B. A. Strietelmeier, andB. J. Travis. 2000. Biological reduction of uranium in groundwater andsubsurface soil. Sci. Total Environ. 250:21–35.

2. Adachi, J., and M. Hasegawa. 1996. MOLPHY version 2.3: programs formolecular phylogenetics based on maximum likelihood. Comput. Sci.Monogr. 28:1–150.

3. Alewell, C., and M. Gehre. 1999. Patterns of stable S isotopes in a forestedcatchment as indicators for biological S turnover. Biogeochemistry 47:319–333.

4. Alewell, C., and A. Giesemann. 1996. Sulfate reduction in a forested catch-ment as indicated by d34S values of sulfate in soil solutions and runoff. Isot.Environ. Health Stud. 32:203–210.

5. Alewell, C., and M. Novak. 2001. Spotting zones of dissimilatory sulfatereduction in a forested catchment: the 34S-35S approach. Environ. Pollut.112:369–377.

6. Alm, E. W., D. B. Oerther, N. Larsen, D. A. Stahl, and L. Raskin. 1996. Theoligonucleotide probe database. Appl. Environ. Microbiol. 62:3557–3559.

7. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.Basic local alignment search tool. J. Mol. Biol. 215:403–410.

8. Bensadoun, J., M. Tonolla, A. Demarta, F. Barja, and R. Peduzzi. 1998.Vertical distribution and microscopic characterization of a non-culturablemicroorganism (morphotype R) of Lake Cadagno. Doc. Ist. Ital. Idrobiol.Dott. Marco Marchi 63:45–51.

9. Benson, D. A., I. Karsch-Mizrachi, D. J. Lipman, J. Ostell, B. A. Rapp, andD. L. Wheeler. 2002. GenBank. Nucleic Acids Res. 30:17–20.

10. Berge, E., J. Bartnicki, K. Olendrzynski, and S. G. Tsyro. 1999. Long-termtrends in emissions and transboundary transport of acidifying air pollution inEurope. J. Environ. Manag. 57:31–50.

11. Blodau, C., C. L. Roehm, and T. R. Moore. 2002. Iron, sulfur, and dissolvedcarbon dynamics in a northern peatland. Arch. Hydrobiol. 154:561–583.

12. Boone, D. R., and M. P. Bryant. 1980. Propionate-degrading bacterium,Syntrophobacter wolinii sp. nov., gen. nov., from methanogenic ecosystems.Appl. Environ. Microbiol. 40:626–632.

13. Brofft, J. E., J. V. McArthur, and L. J. Shimkets. 2002. Recovery of novelbacterial diversity from a forested wetland impacted by reject coal. Environ.Microbiol. 4:764–769.

14. Castro, H., K. R. Reddy, and A. Ogram. 2002. Composition and function ofsulfate-reducing prokaryotes in eutrophic and pristine areas of the FloridaEverglades. Appl. Environ. Microbiol. 68:6129–6137.

15. Chang, B. V., L. C. Shiung, and S. Y. Yuan. 2002. Anaerobic biodegradationof polycyclic aromatic hydrocarbon in soil. Chemosphere 48:717–724.

16. Chang, Y. J., A. D. Peacock, P. E. Long, J. R. Stephen, J. P. McKinley, S. J.Macnaughton, A. K. Hussain, A. M. Saxton, and D. C. White. 2001. Diversityand characterization of sulfate-reducing bacteria in groundwater at a ura-nium mill tailings site. Appl. Environ. Microbiol. 67:3149–3160.

17. Crane, B. R., L. M. Siegel, and E. D. Getzoff. 1995. Sulfite reductase structureat 1.6 A: evolution and catalysis for reduction of inorganic anions. Science270:59–67.

18. Devereux, R., and G. W. Mundfrom. 1994. A phylogenetic tree of 16S rRNAsequences from sulfate-reducing bacteria in a sandy marine sediment. Appl.Environ. Microbiol. 60:3437–3439.

19. Dhillon, A., A. Teske, J. Dillon, D. A. Stahl, and M. L. Sogin. 2003. Molecularcharacterization of sulfate-reducing bacteria in the Guaymas Basin. Appl.Environ. Microbiol. 69:2765–2772.

20. Dunfield, P. F., M. Tchawa Yimga, S. N. Dedysh, U. Berger, W. Liesack, andJ. Heyer. 2002. Isolation of a Methylocystis strain containing a novel pmoA-like gene. FEMS Microbiol. Ecol. 41:17–26.

21. Felsenstein, J. 1995. PHYLIP: phylogeny inference package, version 3.57c.Department of Genetics, University of Washington, Seattle.

22. Friedrich, M. W. 2002. Phylogenetic analysis reveals multiple lateral trans-fers of adenosine-5�-phosphosulfate reductase genes among sulfate-reducingmicroorganisms. J. Bacteriol. 184:278–289.

23. Garrity, G. M., and J. G. Holt. 2001. The road map to the manual, p.119–166. In G. M. Garrity (ed.), Bergey’s manual of systematic bacteriology,2nd ed., vol. 1. Springer, New York, N.Y.

24. Giovannoni, S. J., T. D. Mullins, and K. G. Field. 1995. Microbial diversityin oceanic systems: rRNA approaches to the study of unculturable microbes,p. 217–248. In J. Joint (ed.), Molecular ecology of aquatic microbes, vol. G38.Springer-Verlag, Berlin, Germany.

25. Griffiths, R. I., A. S. Whiteley, A. G. O’Donnell, and M. J. Bailey. 2000. Rapidmethod for coextraction of DNA and RNA from natural environments foranalysis of ribosomal DNA- and rRNA-based microbial community compo-sition. Appl. Environ. Microbiol. 66:5488–5491.

26. Hipp, W. M., A. S. Pott, N. Thum-Schmitz, I. Faath, C. Dahl, and H. G.Truper. 1997. Towards the phylogeny of APS reductases and sirohaem sulfitereductases in sulfate-reducing and sulfur-oxidizing prokaryotes. Microbiol-ogy 143:2891–2902.

27. Hopner, T. 1981. Design and use of a diffusion sampler for interstitial waterfrom fine grained sample. Environ. Technol. Lett. 2:187–196.

28. Horn, M. A., C. Matthies, K. Kusel, A. Schramm, and H. L. Drake. 2003.Hydrogenotrophic methanogenesis by moderately acid-tolerant methano-gens of a methane-emitting acidic peat. Appl. Environ. Microbiol. 69:74–83.

29. Jørgensen, B. B. 1982. Mineralization of organic matter in the sea-bed—therole of sulphate reduction. Nature 296:643–645.

30. Jørgensen, B. B. 1977. The sulfur cycle of a coastal marine sediment (Lim-fjorden, Denmark). Limnol. Oceanogr. 22:814–832.

31. Juretschko, S., A. Loy, A. Lehner, and M. Wagner. 2002. The microbialcommunity composition of a nitrifying-denitrifying activated sludge from anindustrial sewage treatment plant analyzed by the full-cycle rRNA approach.Syst. Appl. Microbiol. 25:84–99.

32. Juretschko, S., G. Timmermann, M. Schmid, K. H. Schleifer, A. Pomme-rening-Roser, H. P. Koops, and M. Wagner. 1998. Combined molecular andconventional analyses of nitrifying bacterium diversity in activated sludge:Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations.Appl. Environ. Microbiol. 64:3042–3051.

33. Kane, M. D., L. K. Poulsen, and D. A. Stahl. 1993. Monitoring the enrich-ment and isolation of sulfate-reducing bacteria by using oligonucleotide



http://aem.asm

.org/D

ownloaded from

http://aem.asm.org/

hybridization probes designed from environmentally derived 16S rRNA se-quences. Appl. Environ. Microbiol. 59:682–686.

34. Klein, M., M. Friedrich, A. J. Roger, P. Hugenholtz, S. Fishbain, H. Abicht,L. L. Blackall, D. A. Stahl, and M. Wagner. 2001. Multiple lateral transfersof dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes. J. Bacteriol. 183:6028–6035.

35. Kovarova, M., and P. Draber. 2000. New specificity and yield enhancer ofpolymerase chain reactions. Nucleic Acids Res. 28:E70.

36. Kusel, K., and C. Alewell. 2004. Riparian zones in a forested catchment: hotspots for microbial reductive processes. Ecol. Stud. 172:377–395.

37. Kusel, K., and H. L. Drake. 1995. Effect of environmental parameters on theformation and turnover of acetate in forest soils. Appl. Environ. Microbiol.61:3667–3675.

38. Kusel, K., H. C. Pinkart, H. L. Drake, and R. Devereux. 1999. Acetogenicand sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cellsof the sea grass Halodule wrightii. Appl. Environ. Microbiol. 65:5117–5123.

39. Kusel, K., U. Roth, T. Trinkwalter, and S. Peiffer. 2001. Effect of pH on theanaerobic microbial cycling of sulfur in mining-impacted freshwater lakesediments. Environ. Exp. Bot. 46:213–223.

40. Li, J.-H., K. J. Purdy, S. Takii, and H. Hayashi. 1999. Seasonal changes inribosomal RNA of sulfate-reducing bacteria and sulfate reducing activity ina freshwater lake sediment. FEMS Microbiol. Ecol. 28:31–39.

41. Llobet-Brossa, E., R. Rossello-Mora, and R. Amann. 1998. Microbial com-munity composition of Wadden Sea sediments as revealed by fluorescence insitu hybridization. Appl. Environ. Microbiol. 64:2691–2696.

42. Lovley, D. R., and M. J. Klug. 1983. Sulfate reducers can outcompete meth-anogens at freshwater sulfate concentrations. Appl. Environ. Microbiol. 45:187–192.

43. Loy, A., M. Horn, and M. Wagner. 2003. probeBase: an online resource forrRNA-targeted oligonucleotide probes. Nucleic Acids Res. 31:514–516.

44. Loy, A., A. Lehner, N. Lee, J. Adamczyk, H. Meier, J. Ernst, K.-H. Schleifer,and M. Wagner. 2002. Oligonucleotide microarray for 16S rRNA gene-baseddetection of all recognized lineages of sulfate-reducing prokaryotes in theenvironment. Appl. Environ. Microbiol. 68:5064–5081.

45. Ludwig, W., O. Strunk, S. Klugbauer, N. Klugbauer, M. Weizenegger,J. Neumaier, M. Bachleitner, and K. H. Schleifer. 1998. Bacterial phylogenybased on comparative sequence analysis. Electrophoresis 19:554–568.

46. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A.Buchner, T. Lai, S. Steppi, G. Jobb, W. Forster, I. Brettske, S. Gerber, A. W.Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Konig, T. Liss, R.Lussmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N.Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K. H. Schleifer.2004. ARB: a software environment for sequence data. Nucleic Acids Res.32:1363–1371.

47. Mohn, W. W., and K. J. Kennedy. 1992. Reductive dehalogenation of chlo-rophenols by Desulfomonile tiedjei DCB-1. Appl. Environ. Microbiol. 58:1367–1370.

48. Mohn, W. W., and J. M. Tiedje. 1992. Microbial reductive dehalogenation.Microbiol. Rev. 56:482–507.

49. Noh, S. L., J. M. Choi, Y. J. An, S. S. Park, and K. S. Cho. 2003. Anaerobicbiodegradation of toluene coupled to sulfate reduction in oil-contaminatedsoils: optimum environmental conditions for field applications. J. Environ.Sci. Health Part A 38:1087–1097.

50. Novak, M., R. K. Wieder, and W. R. Schell. 1994. Sulfur during early di-agenesis in sphagnum peat: insights from delta34S ratio profiles in 210Pb-dated peat cores. Limnol. Oceanogr. 39:1172–1185.

51. Oude Elferink, S. J., W. M. Akkermans-van Vliet, J. J. Bogte, and A. J.Stams. 1999. Desulfobacca acetoxidans gen. nov., sp. nov., a novel acetate-degrading sulfate reducer isolated from sulfidogenic granular sludge. Int. J.Syst. Bacteriol. 49:345–350.

52. Pedersen, K., J. Arlinger, L. Hallbeck, and C. Pettersson. 1996. Investiga-tions of subterranean bacteria in deep crystalline bedrock and their impor-tance for the disposal of nuclear waste. Can. J. Microbiol. 42:382–391.

53. Purdy, K. J., D. B. Nedwell, and T. M. Embley. 2003. Analysis of thesulfate-reducing bacterial and methanogenic archaeal populations in con-trasting Antarctic sediments. Appl. Environ. Microbiol. 69:3181–3191.

54. Purkhold, U., A. Pommering-Roser, S. Juretschko, M. C. Schmid, H.-P.Koops, and M. Wagner. 2000. Phylogeny of all recognized species of ammo-nia oxidizers based on comparative 16S rRNA and amoA sequence analysis:implications for molecular diversity surveys. Appl. Environ. Microbiol. 66:5368–5382.

55. Rabus, R., T. Hansen, and F. Widdel. 2000. Dissimilatory sulfate- andsulfur-reducing prokaryotes. In M. Dworkin, S. Falkow, E. Rosenberg,K.-H. Schleifer, and E. Stackebrandt (ed.), The prokaryotes: an evolving

electronic resource for the microbiological community, 3rd ed. [Online.]Springer-Verlag, New York, N.Y.

56. Ravenschlag, K., K. Sahm, and R. Amann. 2001. Quantitative molecularanalysis of the microbial community in marine arctic sediments (Svalbard).Appl. Environ. Microbiol. 67:387–395.

57. Sahm, K., C. Knoblauch, and R. Amann. 1999. Phylogenetic affiliation andquantification of psychrophilic sulfate-reducing isolates in marine arctic sed-iments. Appl. Environ. Microbiol. 65:3976–3981.

58. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

59. Sass, H., H. Cypionka, and H.-D. Babenzien. 1997. Vertical distribution ofsulfate-reducing bacteria at the oxic-anoxic interface in sediments of theoligotrophic Lake Stechlin. FEMS Microbiol. Ecol. 22:245–255.

60. Sass, H., E. Wieringa, H. Cypionka, H.-D. Babenzien, and J. Overmann.1998. High genetic and physiological diversity of sulfate-reducing bacteriaisolated from an oligotrophic lake sediment. Arch. Microbiol. 170:243–251.

61. Scheid, D., and S. Stubner. 2001. Structure and diversity of Gram-negativesulfate-reducing bacteria on rice roots. FEMS Microbiol. Ecol. 36:175–183.

62. Singleton, D. R., M. A. Furlong, S. L. Rathbun, and W. B. Whitman. 2001.Quantitative comparisons of 16S rRNA gene sequence libraries from envi-ronmental samples. Appl. Environ. Microbiol. 67:4374–4376.

63. Sizova, M. V., N. S. Panikov, T. P. Tourova, and P. W. Flanagan. 2003.Isolation and characterization of oligotrophic acido-tolerant methanogenicconsortia from a Sphagnum peat bog. FEMS Microbiol. Ecol. 45:301–315.

64. Smith, S. W., R. Overbeek, C. R. Woese, W. Gilbert, and P. M. Gillevet. 1994.The Genetic Data Environment (GDE): an expandable graphic interface formanipulating molecular information. Comput. Appl. Biol. Sci. 10:671–675.

65. Stackebrandt, E., and B. M. Goebel. 1994. Taxonomic note: a place forDNA-DNA reassociation and 16S rRNA sequence analysis in the presentspecies definition in bacteriology. Int. J. Syst. Bacteriol. 44:846–849.

66. Stahl, D. A., S. Fishbain, M. Klein, B. J. Baker, and M. Wagner. 2002.Origins and diversification of sulfate-respiring microorganisms. AntonieLeeuwenhoek 81:189–195.

67. Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet max-imum likelihood method for reconstructing tree topologies. Mol. Biol. Evol.13:964–969.

68. Stubner, S., and K. Meuser. 2000. Detection of Desulfotomaculum in anItalian rice paddy soil by 16S ribosomal nucleic acid analyses. FEMS Micro-biol. Ecol. 34:73–80.

69. Sun, B., J. R. Cole, and J. M. Tiedje. 2001. Desulfomonile limimaris sp. nov.,an anaerobic dehalogenating bacterium from marine sediments. Int. J. Syst.Evol. Microbiol. 51:365–371.

70. Tchawa Yimga, M., P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack. 2003.Wide distribution of a novel pmoA-like gene copy among type II meth-anotrophs, and its expression in Methylocystis strain SC2. Appl. Environ.Microbiol. 69:5593–5602.

71. Telang, A. J., G. Voordouw, S. Ebert, N. Sifeldeen, J. M. Foght, P. M.Fedorak, and D. W. Westlake. 1994. Characterization of the diversity ofsulfate-reducing bacteria in soil and mining waste water environments bynucleic acid hybridization techniques. Can. J. Microbiol. 40:955–964.

72. Teske, A., C. Wawer, G. Muyzer, and N. B. Ramsing. 1996. Distribution ofsulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) asevaluated by most-probable-number counts and denaturing gradient gelelectrophoresis of PCR-amplified ribosomal DNA fragments. Appl. Environ.Microbiol. 62:1405–1415.

73. Ulrich, G. A., L. R. Krumholz, and J. M. Suflita. 1997. A rapid and simplemethod for estimating sulfate reduction activity and quantifying inorganicsulfides. Appl. Environ. Microbiol. 63:1627–1630.

74. Wagner, M., A. J. Roger, J. L. Flax, G. A. Brusseau, and D. A. Stahl. 1998.Phylogeny of dissimilatory sulfite reductases supports an early origin ofsulfate respiration. J. Bacteriol. 180:2975–2982.

75. Wallrabenstein, C., E. Hauschild, and B. Schink. 1994. Pure culture andcytological properties of Syntrophobacter wolinii. FEMS Microbiol. Lett. 123:249–254.

76. Wind, T., and R. Conrad. 1995. Sulfur compounds, potential turnover ofsulfate and thiosulfate, and numbers of sulfate-reducing bacteria in plantedand unplanted paddy soil. FEMS Microbiol. Ecol. 18:257–266.

77. Wind, T., S. Stubner, and R. Conrad. 1999. Sulfate-reducing bacteria in ricefield soil and on rice roots. Syst. Appl. Microbiol. 22:269–279.

78. Yang, Z., R. Nielsen, N. Goldman, and A. M. Pedersen. 2000. Codon-sub-stitution models for heterogeneous selection pressure at amino acid sites.Genetics 155:431–449.

79. Yavitt, J. B., G. E. Lang, and R. K. Wieder. 1987. Control of carbon miner-alization to CH4 and CO2 in anaerobic, sphagnum derived peat from BigRun, West Virginia. Biogeochemistry 4:141–157.



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