Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses

FEMS Microbiology Ecology 34 (2000) 73^80

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Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses Stephan Stubner *, Katja Meuser Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-StraMe, D-35043 Marburg, Germany Received 24 March 2000; received in revised form 17 August 2000; accepted 21 August 2000

Abstract Two specific primers were developed for the amplification of 16S rRNA genes of Desulfotomaculum lineage 1 to detect members of the genus Desulfotomaculum in rice field soil. The combination of both primers in PCR allowed the specific amplification and cloning of ten 16S rDNA sequences of this group from rice paddy soil DNA extracts. The phylogenetic analysis showed that these sequences formed a deeply branching cluster within Desulfotomaculum lineage 1, together with two sequences from the database and two sequences from a hydrocarbon-contaminated aquifer. Dissimilarity values to validly described species, including recently isolated strains of Desulfotomaculum from rice paddy microcosms, were higher than 12%. Within the new cluster the cloned sequences formed three separate groups which were each represented by at least two sequences with identities of v99% while one sequence represented an additional group. The sequences should represent sulfate-reducing organisms because they clearly fell into the physiologically coherent group of Gram-positive sulfate reducers. The relative abundance of bacteria of the Desulfotomaculum lineage 1 in rice paddy soil and root samples was estimated with rRNA dot blot hybridizations of extracted RNA. The relative RNA content of Desulfotomaculum lineage 1 was 0.55% in the bulk soil and 1% in the rice root samples, respectively, of the total 16S rRNA content (probe Eub338). Hybridization of rRNA with a probe targeting the new cluster represented by the cloned sequences confirmed the high abundance of 16S rRNA sequences from this cluster in the rice paddy field samples. Another hybridization probe detecting Desulfotomaculum acetoxidans and two closely related Desulfotomaculum isolates from rice paddy soil indicated that these bacteria were less abundant. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Rice ¢eld soil; Sulfate reduction; 16S rDNA; Speci¢c ampli¢cation; Dot blot hybridization ; Desulfotomaculum lineage 1

1. Introduction Rice paddy soils are mainly anoxic habitats. In the bulk soil, anaerobic processes such as denitri¢cation, ferric iron reduction, sulfate reduction and methanogenesis are the main pathways for the degradation of organic matter [1,2]. Due to generally low concentrations of sulfur compounds in rice paddy ¢elds, sulfate reduction in the bulk soil is limited [3,4]. However, Wind and Conrad [5] measured high sulfate reduction rates in 13 weeks old rice paddy microcosms. This can be explained by the reoxidation of reduced electron acceptors in oxygenated zones such as the upper soil layer and the vicinity of the rice roots [6^8]. This theory of an active S cycle in habitats with oxic/anoxic interfaces is now well established [9^12]. For rice ¢eld soil it is con¢rmed by (i) highest in situ

* Corresponding author. Tel. : +49 (6421) 178 740 ; Fax: +49 (6421) 178 999; E-mail : [email protected]

sulfate reduction rates and (ii) highest cell numbers of sulfate reducers in or near oxygenated zones [3,13,14]. In the vicinity of the rice roots a further stimulating e¡ect on sulfate reducers should be the presence of a variety of easily degradable root exudates of the rice plants [15^ 17]. It can be assumed that these e¡ects will select for `plant-speci¢c' sulfate reducers [14]. These should be mainly fast growing, lactate or H2 utilizing, incompletely oxidizing members of the Desulfovibrionaceae [3,13,14]. In the bulk soil, on the other hand, the most abundant species found were acetate consuming sulfate reducers from the Desulfobacteriaceae, and Gram-positive strains of the genus Desulfotomaculum [13,14]. Spore-forming Gram-positive sulfate-reducing bacteria are classically embraced in the genus Desulfotomaculum. Recently, comparative 16S rDNA sequence analysis showed that this group of organisms is not monophyletic but comprises at least two distinct lineages of descent. Most of the validly described species fall within Desulfotomaculum lineage 1. Desulfotomaculum orientis forms a

0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 7 6 - 3

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separate lineage together with Desul¢tobacterium sp. and has recently been renamed Desulfosporosinus orientis. Finally, Desulfotomaculum guttoideum clusters closely to various Clostridia and probably represents a misidenti¢ed species [18,19]. Desulfotomaculum is a physiologically diverse group including thermophilic and mesophilic species that consume a variety of carbon sources [19^21]. Due to their capability of forming endospores, they should be well adapted to survive in environments with regularly changing redox conditions, such as rice ¢eld soil, which is drained between the vegetation periods [20]. Indeed four strains of Desulfotomaculum have been enriched and isolated from rice paddy microcosms [14]. However, quantitative data on the abundance of Desulfotomaculum sp. in rice ¢eld soil are lacking. Therefore, in this work a molecular, culture-independent approach was used to gain more insight into the abundance and distribution of species of Desulfotomaculum in rice ¢eld soil. Because the four isolates, obtained from rice ¢eld soil, were related to the Desulfotomaculum lineage 1, this work concentrated on this lineage. 2. Materials and methods 2.1. Environmental samples and bacterial strains Soil and rice root samples (Oryza sativa, type japonica, variety Korall) were taken from paddy ¢elds at the Italian Rice Research Institute, Vercelli, northern Italy. Sampling was in June 1998 and June and August 1999 between the tillering and the £owering phases of the rice plants. Soil characteristics have been reported previously by Schu«tz et al. [22]. The samples were transported at 4³C. The bulk soil samples originated from the paddy ¢eld from a depth of 5^10 cm and a distance of at least 20 cm to the rice plants. The roots of four^six plants were intermixed for one sample, washed with PBS bu¡er until no visible soil particles were left and stored at 320³C until use. Desulfotomaculum strains R-AcetonA170, R-PimA1, RAcA1, R-IbutA1, Desulfotomaculum thermobenzoicum, Desulfovibrio strain R-SucA1, Desulfobulbus strain RPropA1, Thiobacillus thioparus strain TBW3 and Clostridium sporosphaeroides were used as references for the determination of stringencies for PCR and hybridization. These strains were cultured as described previously [14,23] or as described by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). 2.2. Extraction of nucleic acids Extraction of nucleic acids was done according to the protocol from Lu«demann et al. [24]. The protocol included two cycles of beat beating, the second with a phenol saturated extraction bu¡er for complete lysis of bacterial

cells. For RNA extraction the puri¢cation included phenol/chloroform/isoamylalcohol (25:24:1, v/v/v) extraction followed by two cycles of chloroform/isoamylalcohol (24:1, v/v) extraction. DNA extraction included three cycles of chloroform/isoamylalcohol extraction. The nucleic acids were precipitated with ethanol and resuspended in ultra pure water (Sigma Aldrich GmbH, Deisenhofen, Germany). For RNA extraction, the DNA was removed with RQ1-RNase free DNAse (Promega, Mannheim, Germany) for 30 min at 37³C followed by another chloroform/isoamylalcohol extraction and precipitation. Quanti¢cation of rRNA was done by dot blot hybridization with probe Eub338 using an Escherichia coli 16S rRNA standard (E. coli MRE600 16S and 23S ribosomal RNA, Roche Diagnostics, Mannheim, Germany) in dilution series as described below. 2.3. In vitro transcription No cultures of organisms as controls were available for the determination of the stringency of probe DEMRC1015r in rRNA dot blot hybridization. Therefore, the in vitro transcripts of the cloned 16S rDNA sequence of clone DEM-KMe98-9 (0 mismatches) were used. In vitro transcription was done with the Riboprobe Combination System and with the T7-RNA polymerase according to the protocol of the manufacturer (Promega, Mannheim, Germany). Puri¢cation of the transcripts was done with RNeasy Mini1 kit (Qiagen, Hilden, Germany). 2.4. PCR ampli¢cation Ampli¢cation of 16S rDNA was carried out in the Primus cycler (MWG Biotech, Ebersberg, Germany) and the Gene Amp PCR system 2400 (PE Biosystems, Weiterstadt, Germany). The reaction mixture (100 Wl) contained 1 Wl template DNA, 10 Wl 10Ureaction bu¡er (PCR bu¡er II, PE Biosystems), 150 nmol MgCl2 , 20 nmol of each deoxynucleotide (NucleicPlus, Amersham Pharmacia Biotech, Braunschweig, Germany), 30 pmol of each primer (MWG Biotech, Ebersberg, Germany) and 2.5 U of Taq DNA polymerase (PE Biosystems). The thermal pro¢les included 30 to 35 cycles of primer annealing for 1 min (primerspeci¢c temperature), primer extension at 72³C for 2 min and denaturing at 94³C for 1 min. A ¢nal cycle was done with primer annealing (1 min) and primer extension for 7 min. The ampli¢cation of 16S rDNA from environmental samples was carried out with the primers 9/27f and 1492/1512r [25]. The PCR products were puri¢ed with Qiaquick1 columns according to the manufacturer (Qiagen GmbH, Hilden, Germany). The PCR products were then used for ampli¢cation with primers speci¢c for 16S rDNA of Desulfotomaculum sp. The primers and probes used are shown in Table 1. For determination of the stringency of the annealing temperature of PCR with speci¢c primers, the 16S rDNA PCR products of the reference

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organisms were used. Equal amounts of PCR products (0.15 ng per assay) from positive (0 mismatches) and negative (one to three mismatches) controls were used in PCR assays with increasing annealing temperature. Annealing was assumed to be speci¢c, when the positive controls but not the negative controls yielded a PCR product of the expected fragment size as visualized on agarose gels. 2.5. Cloning and sequencing Ampli¢ed 16S rDNA PCR products were cloned using the pGEM0 -T cloning kit with the supplied vector and E. coli JM109 competent cells according to the manufacturer (Promega, Mannheim, Germany). DNA of randomly selected clones was isolated and puri¢ed according to Rotthauwe et al. [26]. Sequencing was done with the ABI Prism1 Dye Terminator Ready Reaction kit on a 373 DNA sequencer (PE Applied Biosystems, Weiterstadt, Germany) with primers described by Lane [27] and Weisburg et al. [25]. 2.6. Dot blot hybridization The rRNA samples from bacterial isolates, the in vitro transcript and environmental rRNA samples were denatured in 7.3% (v/v) formaldehyde and 6USSC at 68³C for 15 min [28], transferred to positively charged nylon membranes (Hybond1-N+, Amersham Pharmacia Biotech, Braunschweig, Germany) with a vacuum blotter (Bio Dot SF, Bio-Rad, Mu«nchen, Germany) and UV crosslinked (Stratalinker0 2400, Stratagene, USA). Hybridization with DIG-labeled probes (MWG Biotech, Ebersberg, Germany) was done according to the protocol and bu¡ers recommended by Roche (Roche diagnostics GmbH, Mannheim, Germany). Prehybridization was done for 1^2 h at 40³C. Hybridization was performed overnight at 38³C or 40³C with 1.5 pmol oligonucleotide probe per cm2 membrane. The membranes were washed at 40³C four

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times in 0.1^5USSC and 0.1% (w/v) sodium dodecyl sulfate. Detection of the DIG dye was performed according to the manufacturer (DIG Nucleic Acid Detection Kit, Roche diagnostics GmbH, Mannheim, Germany). The dilution of the anti-DIG antibody alkaline phosphatase conjugate was 1:1000 (v/v) in blocking bu¡er. The signals were visualized on a £uorescence scanner (Storm1860, Amersham Pharmacia Biotech, Braunschweig, Germany), after addition of the chemiluminescence substrate ECF1 (Amersham Pharmacia Biotech) and incubation for 20 min in the dark. Quanti¢cation was carried out with the program Image Quant (Molecular Dynamics, USA). Dilution series of rRNAs from reference organisms were included on each membrane for standardization. The stringency for washing after the hybridization was determined with RNA from pure cultures, or with the in vitro RNA transcripts in various dilutions (108 ^5U1010 targets per slot) for each probe (see also Table 1 and Section 3). 2.7. Phylogenetic analysis The ARB software package [29] together with the database from December 1998 was used for primer and probe design, sequence alignment, comparison and construction of phylogenetic trees. The sequences of Desulfotomaculum luziae and Desulfotomaculum alkaliphilum were obtained from the EMBL database and integrated into the ARB program. Primers and probes were designed using the probe design and probe match tools. Phylogenetic trees were constructed using maximum likelihood and neighbor-joining methods provided in the program. To omit highly variable regions, various ¢lters for Desulfotomaculum lineage 1 were used with 40 and 50% invariance, respectively. Chimeric rDNA sequences were identi¢ed by separated sequence comparison of the 5P and 3P terminal 300 base pairs. Three sequences of Moorella sp., Thermoterrabacterium sp. and Thermoanaerobacter sp. were used as outgroup references.

Table 1 Oligonucleotide primers and hybridization probes Primer/probe

Sequence

Target group

9/27f 1492/1512ra Eub338b DEM116fa

GAGTTT G(A/C)TCCT GGCTCA G ACGG(C/T)T ACCTTG TTACGA CTT GCTGCC TCCCGT AGGAGT GTAACG CGTGGA TAACCT

eubacteria eubacteria eubacteria Desulfotomaculum lineage 1

DEM1164ra;b

CCTTCC TCCGTT TTGTCA

Desulfotomaculum lineage 1

D-acet1027rb

CTCCGT GTGCAA GTAAAC

DEM-RC1015rb

GAAGCT GGAAAA CGCACT

D. acetoxidans, R-AcA1, R-IbutA1 cluster of sequences from rice paddy soil

a

Organisms for speci¢city testsc

R-AcetonA1 (0), R-PimA1 (0), R-SucA1 (1), TBW3 (2) R-AcetonA1 (0), R-PimA1 (0), TBW3 (1), R-SucA1 (2), R-PropA1 (3) R-AcA1 (0) clone sequence DEM-Kme98-9 (0), D. thermobenzoicum (4)

Reference [25] [25] [37] this study this study this study this study

A description of the target groups of the Desulfotomaculum sp. speci¢c oligonucleotides is given in Section 3. a PCR primer. b Probe for dot blot hybridization. c 16S rDNA or 16S rRNA of these organisms (in vitro transcript of clone sequence DEM-Kme98-9) were used as positive and negative controls for speci¢city tests of PCR and dot blot hybridization. The number of mismatches in the respective primer-binding sites is given in brackets.

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The sequences were submitted to the EMBL database under the accession numbers AJ276557^AJ276566. 3. Results 3.1. Design and evaluation of group-speci¢c primers For the speci¢c ampli¢cation of the 16S rDNA of members of the Desulfotomaculum lineage 1 sensu Stackebrandt et al. [18], two primers were developed using the probe design and probe match tool of the ARB software package (Table 1). Primer DEM1164r binds to positions 1164^1182 of the 16S rDNA (numbering according to Brosius et al. [30]) and targets the complete Desulfotomaculum lineage 1 without mismatch (D. alkaliphilum and Desulfotomaculum halophilum have one mismatch at position 1181). A few non-Desulfotomaculum sequences matched the target region of the primer. These included several strains of Ruminococcus sp., two strains of Eubacterium sp. and one sequence of Nitrosococcus sp., Clostridium sp. and Fusobacterium sp., respectively. 16S rDNA sequences with one mismatch in the primer-binding site were distributed over a wide range of bacteria. All these sequences showed the same mismatch in the target region (C instead of A at position 1172). The primer DEM1164r was used in PCR together with the general eubacterial primer 9/27f. Speci¢c PCR was achieved at an annealing temperature of 65³C, as tested with 16S rDNA PCR products of positive and negative controls (not shown). The second primer speci¢c for members of the genus Desulfotomaculum, DEM116f, binds to positions 116^133 of the 16S rDNA. It targets most members of Desulfotomaculum lineage 1. The Desulfotomaculum strains R-AcA1 and R-IbutA1 (both isolated from rice ¢eld soil), D. acetoxidans and one strain of Desulfotomaculum ruminis showed one mismatch in the primer-binding site. Several non-related organisms, including species related to Clostridium, Heliobacterium, Eubacterium and Desul¢tobacterium, had 16S rDNA sequences that matched the primer sequence. The database showed many organisms with one mismatch in the primer region, including many species of Clostridium sp., Eubacterium sp., various N-proteobacteria and others. Stringent PCR conditions were again established (primers DEM116f, 1492/1512r) using 16S rDNA of reference organisms with 0^two mismatches. Stringent ampli¢cation was possible at an annealing temperature of 68³C (not shown). Because of the better speci¢city of the primer DEM1164r, a ¢rst clone library was constructed with this primer. DNA was extracted from bulk soil and root samples from Italian rice ¢elds and the 16S rDNA was ampli¢ed with the primers 9/27f and 1492/1512r. The puri¢ed PCR products were ampli¢ed with 9/27f and DEM1164r and the products were cloned. Ten cloned

Fig. 1. Speci¢c PCR conditions for the ampli¢cation of 16S rDNA of Desulfotomaculum lineage 1 organisms with the primer combination DEM116f and DEM1164b. General 16S rDNA PCR products from different bacterial strains were used as templates for PCR as described in Section 2. The annealing temperature was 63³C. Lanes 1 and 11, DNA fragment size standard (smart ladder, Eurogentech, Belgium) ; lane 2, Desulfotomaculum sp. R-PimA1 (0 MM); lane 3, Desulfotomaculum sp. R-AcetonA170 (0 MM); lane 4, Desulfotomaculum sp. R-AcA1 (1 MM); lane 5, Desulfotomaculum sp. R-IbutA1 (1 MM); lane 6, D. thermobenzoicum (0 MM); lane 7, Desulfobulbus sp. R-Prop A1 (4 MM); lane 8, T. thioparus TBW3 (8 MM); lane 9, C. sporosphaeroides (2 MM); lane 10, negative PCR control.

fragments were partially sequenced and shown to be related to Clostridium sp., Frankia sp. or Eubacterium sp. but not to Desulfotomaculum sp. Therefore, in a second approach the combination of the primer DEM116f together with DEM1164r was used for speci¢c ampli¢cation. The speci¢city of this primer set was again determined by PCR of 16S rDNA products from several organisms possessing various numbers of mismatches in the primer-binding regions. Speci¢c annealing was possible at a temperature of 63³C (Fig. 1). The ampli¢cation under stringent PCR conditions showed a good yield of PCR products from positive controls (Desulfotomaculum strains R-PimA1, R-AcetonA170 and D. thermobenzoicum) and a weaker signal from sequences with one mismatch in the primer-binding region (Desulfotomaculum strains R-AcA1 and R-IbutA1). No ampli¢cation occurred with DNA from C. sporosphaeroides which had two mismatches in the primer-binding regions. With the general PCR products (primers 9/27f, 1492/ 1512r) of the environmental DNA extracts from bulk soil, speci¢c ampli¢cation with the primers DEM116f and DEM1164r gave PCR products which were cloned. Several partial sequences of cloned fragments from three di¡erent bulk soil clone libraries were compared with the EMBL database. Twelve of these were related most closely to Desulfotomaculum sp. Only few sequences of the clone libraries were a¤liated to Frankia sp. or Clostridium sp. rather than Desulfotomaculum sp. No ampli¢cation products could be obtained from DNA extracts of the root samples.

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Fig. 2. Phylogenetic analysis of cloned 16S rDNA Desulfotomaculum sequences from rice bulk soil. The tree was constructed with the neighbor-joining method of the ARB program package and the Jukes^Cantor correction. A ¢lter of 50% invariance for Desulfotomaculum lineage 1 was used to omit highly variable regions within the 16S rDNA sequences. The bar indicates the estimated number of base changes per nucleotide sequence position. As outgroup references, sequences of Moorella sp., Thermoterrabacterium sp. and Thermoanaerobacter sp. were used.

3.2. Phylogenetic analysis of Desulfotomaculum 16S rDNA cloned sequences The sequences of the 12 clone inserts between positions 135 and 1160, according to the E. coli numbering, were

determined. Two of the clone sequences were probably of chimeric origin and were excluded from further analysis. The phylogenetic analysis of the ten clone sequences showed that they formed a new cluster together with the two sequences, Spore A and Spore B, from the database

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and with the environmental sequences, WCHB1-2 and WCHB1-8 [31]. This cluster branched deep within Desulfotomaculum lineage 1 (Fig. 2). The rice ¢eld isolate Desulfotomaculum R-AcetonA170 [14] fell in many phylogenetic analyses within this cluster, albeit at a deep branching point. Similarity values within this cluster were higher than 91%. The Desulfotomaculum strain RAcetonA170 showed 88^90% sequence identity to the sequences of this cluster. Sequence identity of this cluster to other Desulfotomaculum sequences was less than 88%. Five of the clone sequences (clones DEM-KMe98-4, DEMKMe98-5, DEM-KMe98-7, DEM-KMe98-8, DEMKMe98-9) were almost identical having dissimilarity values of 0.6^1.0% to each other and dissimilarities to the closest neighbors of 4.6% (Spore A) and 5.1% (WCHB12), respectively. The two clone sequences DEM-KMe99-1 and DEM-KMe99-3 were 99.6% identical and had 3% dissimilarity to the next neighbor (Spore A). The two clones DEM-KMe98-6 and DEM-KMe98-10 shared 99.4% sequence identity, and had a dissimilarity of more than 6% to other sequences (WCHB1-2 and WCHB1-8). Finally the clone sequence DEM-KMe99-2 had a minimum dissimilarity of 4.2% to other sequences within this cluster. 3.3. Determination of the relative abundance of Desulfotomaculum sp. in bulk soil and rice root samples The abundance of Desulfotomaculum lineage 1 in the rice ¢eld samples was estimated with rRNA-targeted dot blot hybridization using probe DEM1164r. Speci¢city of the hybridization was tested with rRNA of reference organisms (see also Table 1). Stringency was achieved by washing, after the hybridization, at a temperature of 40³C with 0.1USSC. For the determination of the abundance of rRNA from bacteria represented by the cloned sequences, probe DEM-RC1015r was developed. This probe targets eight of the ten cloned sequences and had at least four mismatches to all other sequences of the ARB database. Hybridization conditions were tested with the in vitro transcript of clone DEM-KMe98-9 (positive control) and with the rRNA from D. thermobenzoicum (negative control, four mismatches). Stringent hybridization was possible at a washing temperature of 38³C with 1USSC. A further probe, D-acet1027r, was developed to target D. acetoxidans and the two rice ¢eld isolates Desulfotomaculum strains R-AcA1 and R-IbutA1. This probe has at least four mismatches to other sequences in the database. Stringent hybridization was achieved at a washing temperature of 40³C with 1USSC. Dot blot hybridizations with these probes were performed against rRNA extracted from bulk soil and from rice root samples (Table 2). For quanti¢cation, the hybridization signals were compared to the hybridization signals obtained with the general probe Eub338 (%). The probe DEM1164r showed a relative rRNA content of Desulfoto-

Table 2 Relative 16S rRNA contents of Desulfotomaculum sp. in rice ¢eld samples Probe

Bulk soil

Rice roots

DEM1164r DEM-RC1015r D-acet1027r

0.55% ( þ 0.08) 0.60% ( þ 0.1) 6 0.2%

1.0% ( þ 0.12) 0.9% ( þ 0.2) 6 0.2%

The 16S rRNA content of di¡erent groups of Desulfotomaculum in rice ¢eld samples was determined by dot blot hybridizations. The relative content is given as percentage of the general eubacterial 16S rRNA content, which was obtained via hybridization with probe Eub338.

maculum lineage 1 organisms of 0.55% in bulk soil and 1% at the rice roots. Similar values were obtained using probe DEM-RC1015r. Probe D-acet1027r did not give any hybridization signal from the environmental samples. The relative abundance of the rRNA of this group therefore was less than 0.2% in all samples based on the total amount of rRNA on the blots and the sensitivity threshold for this probe. 4. Discussion The present work aimed to detect bacteria of the genus Desulfotomaculum in rice ¢eld soil using molecular methods. To this end, primers were developed for PCR ampli¢cation of the 16S rDNA of this group. Primer DEM1164r showed good coverage of the Desulfotomaculum lineage 1 with only few exceptions. These included two Desulfotomaculum strains with one mismatch (false negatives) and only few non-target organisms without mismatches (false positives). The stringency tests with primer DEM1164r combined with primer 9/27f of positive and negative controls showed good PCR speci¢city, when equal amounts of the target sequence were used. This was not true for the ampli¢cation assays with environmental DNA extracts that did not yield Desulfotomaculum 16S rDNA sequences. Organisms with one mismatch in the primer-binding region might be much more abundant in rice ¢eld soil than Desulfotomaculum sp. The resulting much higher content of 16S rDNA sequences with one mismatch in the environmental DNA extracts probably led to their ampli¢cation despite the speci¢city of the PCR. In a second approach, PCR was performed using both speci¢c primers for Desulfotomaculum sp., DEM116f and DEM1164r. Combining both speci¢c primers, only one organism, C. sporosphaeroides, had as few as two mismatches (both in DEM1164r). All other non-target sequences in the database had at least three mismatches. Combining the two speci¢c primers therefore led to a higher speci¢city for PCR ampli¢cation. Indeed, this PCR assay allowed the ampli¢cation and cloning of 16S rDNA Desulfotomaculum sequences.

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The phylogenetic analysis showed that the ten cloned Desulfotomaculum sequences fell clearly into Desulfotomaculum lineage 1. Because all cultured members of this lineage are capable of sulfate reduction, the cloned sequences should represent Gram-positive sulfate-reducing bacteria. The sequences formed a deep branching cluster within lineage 1 together with four sequences from the database. The similarities among the sequences within this cluster were signi¢cantly higher than the similarity to any other sequences of the genus Desulfotomaculum, con¢rming the monophyletic origin of these sequences. Within this cluster the cloned sequences of this work showed three groups of sequences with high similarities of over 99%, while clone DEM-KMe99-2 represented one separate group. Strains of the same species should have at least 97.5% similarity of their 16S rDNA sequences [32]. Although the de¢nition of a species should be based mainly on physiological characteristics, the high similarities of the cloned sequences within the groups suggest that they represent no more than four species. Three of these species/groups are represented by more than one sequence with nine of ten sequences representing only three groups within the one deep branching cluster. It can therefore be assumed that these are the predominating Desulfotomaculum sequences in rice bulk soil. This is supported by the results of the rRNA dot blot hybridizations. These hybridizations showed that the relative rRNA content of sequences from this cluster (hybridization probe DEM-RC1015r) approximately equalled the relative rRNA content of the complete Desulfotomaculum lineage 1 (probe DEM1164r). Therefore we are con¢dent that the cloned sequences originate from the most abundant strains of Desulfotomaculum in rice paddy soil and are not caused by PCR artifacts such as preferential ampli¢cation due to varying G+C contents of the sequences, or due to DNA concentration e¡ects in the extracts [33^36]. Sequences related to D. acetoxidans might have been missed with the PCR assay. The test of PCR speci¢city already showed that such sequences (one mismatch in the primer-binding regions) yielded less PCR product. This is problematic because the presence of D. acetoxidans-related strains in rice ¢eld soil has been shown by the isolation of the two strains R-AcA1 and R-IbutA1. Therefore a further hybridization (D-acet1027r) probe was developed, targeting this group. But these organisms could not be detected in bulk soil or rice root samples by rRNA dot blot hybridization. Future work will address the question of the abundances of di¡erent groups of Desulfotomaculum in rice ¢eld soil and their location in correlation to the rice roots in more detail.

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