Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Envi

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 2080–2091 Vol. 72, No. 3 0099-2240/06/$08.00ϩ0 doi:10.1128/AEM.72.3.2080–2091.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Non-Sulfate-Reducing, Syntrophic Bacteria Afﬁliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments

Hiroyuki Imachi,1* Yuji Sekiguchi,1,2 Yoichi Kamagata,1,2 Alexander Loy,3 Yan-Ling Qiu,1,2 Downloaded from Philip Hugenholtz,4 Nobutada Kimura,2 Michael Wagner,3 Akiyoshi Ohashi,1 and Hideki Harada1 Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan1; Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST) Center 6, Tsukuba, Ibaraki 305-8566, Japan2; Department of Microbial Ecology, University of Vienna, A-1090 Vienna, Austria3; and Microbial Ecology Program, DOE Joint Genome Institute, Walnut Creek, California4

Received 16 November 2005/Accepted 27 December 2005 http://aem.asm.org/

The classical perception of members of the gram-positive Desulfotomaculum cluster I as sulfate-reducing bacteria was recently challenged by the isolation of new representatives lacking the ability for anaerobic sulfate respiration. For example, the two described syntrophic propionate-oxidizing species of the genus Pelotomacu- lum form the novel Desulfotomaculum subcluster Ih. In the present study, we applied a polyphasic approach by using cultivation-independent and culturing techniques in order to further characterize the occurrence, abundance, and physiological properties of subcluster Ih bacteria in low-sulfate, methanogenic environments. 16S rRNA (gene)-based cloning, quantitative ﬂuorescence in situ hybridization, and real-time PCR analyses

showed that the subcluster Ih population composed a considerable part of the Desulfotomaculum cluster I on November 11, 2015 by University of Queensland Library community in almost all samples examined. Additionally, five propionate-degrading syntrophic enrichments of subcluster Ih bacteria were successfully established, from one of which the new strain MGP was isolated in coculture with a hydrogenotrophic methanogen. None of the cultures analyzed, including previously described Pelotomaculum species and strain MGP, consumed sulfite, sulfate, or organosulfonates. In accordance with these phenotypic observations, a PCR-based screening for dsrAB (key genes of the sulfate respiration pathway encoding the alpha and beta subunits of the dissimilatory sulfite reductase) of all enrichments/(co)cultures was negative with one exception. Surprisingly, strain MGP contained dsrAB, which were transcribed in the presence and absence of sulfate. Based on these and previous findings, we hypothesize that members of Desulfotomacu- lum subcluster Ih have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction.

Members of Desulfotomaculum cluster I have been generally treated taxonomically as an individual genus, all subcluster known as gram-positive, spore-forming, sulfate-reducing bac- members were until recently described as Desulfotomaculum teria (50, 56) and have frequently been found in various anoxic species due to the absence of sufficient distinguishing pheno- environments, such as sediments, rice paddy soil, human feces, typic and molecular features (50). However, the two recently and anaerobic sludges (for examples, see references 21, 42, 56, described species of the new genus Sporotomaculum lack the 57). The genus Desulfotomaculum includes over 20 validly de- ability to respire anaerobically with sulfate, although they scribed mesophilic and thermophilic species, all of which share are members of Desulfotomaculum subcluster Ib (3, 44). In the ability to oxidize various organic substances, like short- addition, Pelotomaculum thermopropionicum strain SI, rep- chain fatty acids, alcohols, and aromatic compounds with sul- resenting the novel subcluster Ih within Desulfotomaculum fate as a terminal electron acceptor (56). Due to these physi- cluster I (22, 23), is also lacking dissimilatory sulfate-reduc- ological traits, members of Desulfotomaculum cluster I have ing activity (22, 23). Importantly, strain SI grows syntrophi- been considered important in sulfidogenic, anoxic environ- cally with hydrogenotrophic methanogens (22, 23), whereas ments, where they play crucial roles in the degradation of only a few members of the genus Desulfotomaculum are able organic compounds, as well as in the biogeochemical cycling of to grow in syntrophic association with methanogens in the sulfur (56). absence of sulfate (26, 42). The collection of non-sulfate- On the basis of comparative 16S rRNA gene sequence anal- reducing bacteria belonging to Desulfotomaculum subcluster ysis, Desulfotomaculum cluster I was considered to be com- Ih was recently extended by the successful isolation of further prised of seven well-separated subclusters, Ia to Ig (27, 50, 52, syntrophic strains in coculture with hydrogenotrophic methano- 57). Although it has been argued that each subcluster could be gens: the propionate degrader Pelotomaculum schinkii (6, 7, 16) and the two phthalate-degrading strains JT and JI (42a, 43). Furthermore, Lueders et al. (37) reported that Pelotomaculum * Corresponding author. Mailing address: Department of Environ- spp. (i.e., subcluster Ih microbes) were one of the active in situ mental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9642. syntrophic propionate-oxidizing populations in a rice field by Fax: 81-258-47-9642. E-mail: [email protected]. rRNA-based stable-isotope probing.

2080 VOL. 72, 2006 NON-SULFATE-REDUCING SYNTROPHIC DESULFOTOMACULUM CLUSTER I 2081

TABLE 1. Samples from methanogenic environments analyzed in this study

Reactor Sulfate concn Influent Sample Sample operation Treating waste/wastewater in influent CODcra Location no. temp (°C) (mg/liter) (mg/liter) 1 Thermophilic digested sludge TJ 55 Municipal solid waste 20 93,550 Niigata, Japan 2 Thermophilic digested sludge TO 53 Municipal sewage sludge 56 59,000 Osaka, Japan 3 Thermophilic granular sludge TR2 55 Artificial wastewater 157 4,000 Our laboratory 4 Thermophilic granular sludge TD 55 Wastewater from clear liquor 149 12,000 Our laboratory manufacture Downloaded from 5 Mesophilic granular sludge MR1 35 Artificial wastewater 81 2,000 Our laboratory 6 Mesophilic granular sludge MB 35 Wastewater from beer factory 55 3,000 Ibaraki, Japan 7 Mesophilic granular sludge MC 35 Wastewater from sugar factory 58 1,635 Nara, Japan 8 Mesophilic digested sludge MD 35 Municipal sewage sludge 62 57,600 Niigata, Japan 9 Rice paddy soil RP Niigata, Japan

a Chemical oxygen demand as determined using dichromate. http://aem.asm.org/ The degradation of organic matter by Desulfotomaculum Construction of 16S rRNA gene clone libraries. Extraction of DNA from cluster I bacteria in methanogenic environments has been anaerobic environmental samples and enrichment cultures was done according thought to be linked to dissimilatory sulfate reduction (21, 52). to a previously described protocol (38). Before PCR amplification, the extracted DNA was purified with a GENE CLEAN Turbo kit (BIO101, CA). Amplifica- Based on the results of this study and the previous studies tion of 16S rRNA genes was carried out according to the method in a previous mentioned above, we propose that subcluster Ih bacteria have study (48). The Desulfotomaculum cluster I-specific primer pair DEM116f/ adapted to anoxic, low-sulfate conditions and thus constitute a DEM1164r (52) and the universal bacterial primer pair 341F/1490R (40, 55) significant fraction of the Desulfotomaculum cluster I popula- were used for amplification of 16S rRNA genes from environmental samples and tion in methanogenic ecosystems. As a consequence of this enrichments, respectively (52). The PCR products were purified with a Micro- Spin Column (Amersham Biosciences) and subsequently cloned into E. coli using evolutionary process, they have lost the capability for sulfate the TA cloning kit (Novagen). Ten clones were randomly picked from each clone on November 11, 2015 by University of Queensland Library respiration and live in close proximity to hydrogen- and for- library for sequencing. 16S rRNA gene sequences with a sequence similarity of mate-consuming methanogens in order to maintain an ener- 100% were grouped into a “phylotype.” The name of each phylotype is composed getically favorable, low-hydrogen partial pressure for the syn- of the sample name and a number (for example, TJ-1 is clone number 1 recovered from the environmental sample TJ). trophic oxidation of organic substrates. PCR amplification of dsrAB and apsA. DNA extractions from pure (co)cultures (P. thermopropionicum, P. schinkii, S. fumaroxidans, and strains JT, JI, and MATERIALS AND METHODS MGP), as well as from enrichment cultures containing subcluster Ih bacteria, were performed according to the method of Hiraishi (20). The template quality Microorganisms and cultivation media. The following pure (co)cultures were of extracted DNA was tested by PCR with the 16S rRNA gene-specific primer used in this study. Pelotomaculum thermopropionicum strain SI was isolated in pair 341F (40) and 907R (28). PCR amplifications of the dsrAB and apsA genes our previous study (22, 23). Strain MGP was isolated in this study as an anaer- were performed as reported previously (9, 14, 25). Approximately 40 ng of obic, mesophilic, syntrophic propionate-oxidizing bacterium. Methanosaeta ther- template DNA was used in a 100-␮l PCR mixture; the concentration of template mophila strain PT (DSM 6194), Methanothermobacter thermautotrophicus strain DNA was measured by using the PicoGreen double-stranded DNA quanti- ⌬H (DSM 1053), Methanospirillum hungatei strain JF1 (DSM 864), Methanosaeta fication kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; concilii strain GP6 (DSM 3671), Methanobacterium bryantii strain M.o.H (DSM Shimadzu). An approximately 1,900-bp fragment of dsrAB was amplified by using 863), Syntrophobacter fumaroxidans strain MPOB (DSM 10017), Desulfotomacu- lum thermobenzoicum strain TSB (DSM 6193), Desulfotomaculum thermo- the primers DSR1Fdeg and DSR4Rdeg (25). The primer pairs APS-FW/ sapovorans strain MLF (DSM 6562), and Desulfotomaculum thermocisternum APS-RV (9) and APS7-F (and its derivatives)/APS8-R (14) were used for am- strain ST90 (DSM 10259) were obtained from the Deutsche Sammlung von plification of approximately 390- and 900-bp fragments of apsA, respectively. Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Moorella Genomic DNA from S. fumaroxidans was used as the positive control in all thermoacetica (JCM 9319) was obtained from the Japan Collection of Microor- dsrAB- and apsA-targeted PCR experiments (14). PCR products of dsrAB and ganisms (Wako, Japan). Eschericha coli strain JM109 was purchased from apsA were purified with a MicroSpin Column (Amersham Biosciences) and TaKaRa Bio Inc. (Otsu, Shiga, Japan). Pelotomaculum schinkii strain HH was either directly sequenced or cloned in E. coli with the TA cloning kit (Novagen) kindly provided by Frank de Bok (Wageningen University, Wageningen, The prior to being sequenced. Netherlands). In addition, we also used the following microorganisms isolated in Sequencing and phylogenetic analysis. The complete 16S rRNA gene se- our laboratory: an anaerobic, syntrophic terephthalate-degrading bacterium, quence of strain MGP isolated in this study was determined as described previ- strain JT (43); an anaerobic, syntrophic isophtalate-degrading bacterium, strain ously (23). The sequences of the 16S rRNA, dsrAB, and apsA genes were deter- JI (Qiu et al., unpublished); and a hydrogen- and formate-utilizing methanogen, mined by dye terminator cycle sequencing with a Quick Start kit (Beckman Methanothermobacter thermautotrophicus strain type II. The culture media used Coulter) and an automated sequence analyzer (CEQ2000XL; Beckman Coulter). If for the enrichment and cultivation of the above-mentioned reference microor- not otherwise indicated, phylogenetic analyses were performed with the ARB ganisms were prepared as described previously (47). All cultivations were carried program (36). A 16S rRNAgene-based tree was constructed by applying the out at either 55°C or 37°C in 50-ml serum vials containing 20 ml of medium (pH neighbor-joining method (45) for sequences having Ͼ1,000 nucleotides. Subse- at 25°C, 7.2) under an atmosphere of N2-CO2 (80/20 [vol/vol]) without shaking. quently, shorter sequences were inserted into this tree without changing the tree The purity of strain MGP was routinely examined by microscopy and incubation topology by using the parsimony insertion tool of the ARB program. Bootstrap of the cultures with medium containing 10 mM sucrose, 10 mM glucose, 10 mM analysis (1,000 replicates) (12) was performed with the PAUP* 4.0 program lactate, and 10 mM pyruvate plus 0.1% yeast extract, thioglycolate medium, and package (53) to estimate the confidence of the 16S rRNA gene tree topology. AC medium (Difco) at 37 and 55°C. New dsrAB and apsA sequences were aligned and phylogenetically analyzed as Environmental samples. Eight anaerobic-sludge samples and one sample from described previously (14, 35). a rice paddy soil were collected and analyzed in this study (Table 1). The FISH. The following 16S rRNA-targeted oligonucleotide probes were used in operational conditions for two laboratory-scale upflow anaerobic-sludge blanket this study: TGP690 for P. thermopropionicum strain SI (23); EUB338, targeting reactors treating artificial wastewater (environmental TR2 and MR1) were de- most Bacteria (1); and DEM1164r for almost all members of Desulfotomaculum scribed previously (46, 48). All the engineered and environmental samples dis- cluster I (52). A novel 16S rRNA-targeted oligonucleotide probe, Ih820 (5Ј-ACC played methanogenic activity. TCCTACACCTAGCAC-3Ј; 820 to 837 in E. coli positions), targeting members 2082 IMACHI ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 2. 16S rRNA gene-targeted primers used for quantitative real-time PCR

Fragment Mg2ϩ concn Annealing Primer set Target Positive control length (bp) (mM) temp (°C) DEM116f-DEM1164r Desulfotomaculum cluster I P. thermopropionicum 1,054 3 60 DEM116f-Ih820 Desulfotomaculum subcluster Ih P. thermopropionicum 706 3 65 519f-907r Bacteria E. coli 407 4 50 Ar109f-Ar912r Archaea M. bryantii 788 3 52 Downloaded from

of Desulfotomaculum subcluster Ih, was designed by using the ARB program. respectively. PCR products obtained by the RT-PCR experiment were cloned Probe Ih820 was applied with an unlabeled competitor probe (5Ј-ACCTCC with the TA cloning kit (Novagen) and sequenced. TACACCTAGTAC-3Ј; 820 to 837 in E. coli positions) in order to avoid cross- Analytical methods. Short-chain fatty acids, alcohols, methane, hydrogen, car- hybridization with nontarget microorganisms. Further details of the cyanine 3.18 bon dioxide, and other intermediate substances, such as succinate, fumarate, and (Cy3)-labeled probes used in this study are available at probeBase (34). Fixation lactate, were measured as described previously (22, 23). Terephthalate was de- of cells in the environmental samples and enrichment cultures and whole-cell in termined by high-performance liquid chromatography using an Aspak DE413 situ hybridization were performed as described previously (45). Granular-sludge column (Shodex; reverse phase) held at 50°C and a UV detector (Shimadzu) set http://aem.asm.org/ samples TR2, TD, MR1, MB, and MC were dispersed by a manually operated at 230 nm. The mobile phase was a 40:60 (vol/vol) mixture of methanol and 1% homogenizer and an ultrasonic homogenizer prior to fluorescence in situ hybrid- aqueous acetic acid at a flow rate of 0.8 ml per min. Sulfate and sulfite were ization (FISH). The stringency of hybridization was adjusted by adding formam- determined by ion chromatography (column, Shimadzu Shim-pack IC-A1; car- ide to the hybridization buffer (15% [vol/vol] for TGP690, 20% for EUB338, 10% rier, 25 mM phthalate hydrogen potassium; detector type, electrical conductivity for DEM1164r, and 20% for Ih820). Enumeration of probe-stained cells was detector). performed with an epifluorescence microscope (Olympus BX50F) as described Nucleotide sequence accession numbers. Sequence data obtained in this study previously (22). were deposited in the DDBJ/EMBL/GenBank databases under accession num- Real-time PCR quantification. Quantitative PCR was performed with a Light- bers AB154373 to AB154392. Cycler device (Roche, Mannheim, Germany) by using the TaKaRa Ex Taq

R-PCR version (Takara Bio Inc., Otsu, Japan) with SYBR Green I (BioWhit- on November 11, 2015 by University of Queensland Library taker Molecular Applications, Rockland, ME). A reaction mixture for PCR was RESULTS prepared according to the manufacturer’s instructions, including approximately 0.1 or 1 ng of template DNA and 0.0006% (vol/vol) SYBR Green I solution. The Phylotype richness of Desulfotomaculum cluster I bacteria. following 16S rRNA gene-targeted PCR primer sets were used: 519f and 907r To extend our current knowledge of the diversity of bacteria of (28, 51) for the domain Bacteria, Ar109f (5Ј-AMDGCTCAGTAACACGT-3Ј;a Desulfotomaculum cluster I in anaerobic environments, sepa- ␤ slightly modified version of a primer reported by Gro kopf et al. [15]) and rate 16S rRNA gene clone libraries were established for nine Ar912r (15) for the domain Archaea, DEM116f and DEM1164r for members of Desulfotomaculum cluster I (51, 52), and DEM116f and Ih820 for Desulfo- methanogenic samples (Table 1) by using a cluster I-specific tomaculum subcluster Ih bacteria. For the construction of template standards for primer pair (52). Ten clones from each library were randomly each primer set, we used dilution series of 16S rRNA gene PCR products of selected, sequenced, and phylogenetically analyzed (Fig. 1). P. thermopropionicum, E. coli, and M. bryantii (obtained with the bacterial primer Most clones (76%) belonged to Desulfotomaculum subcluster pair 8F/1490R [55] or the archaeal primer pair Arch21F/1490R [8]). These PCR Ih and were classified into 16 different phylotypes. The remain- products were used in each real-time PCR analysis to calculate the copy number of 16S rRNA genes in the sample of interest. Template DNA was purified with ing 22 clones formed three phylotypes, which were affiliated a GENE CLEAN Turbo kit (BIO101, CA) and quantified spectrofluorometri- with two other Desulfotomaculum I subclusters. In more detail, cally by using the PicoGreen double-stranded DNA Quantification kit (Molec- 10 clones each from anaerobic reactors treating brewery and ular Probes). The optimal PCR conditions were determined by changing the sugar-manufacturing wastewater were grouped in phylotypes Mg2ϩ concentration in the PCR buffer and the PCR annealing temperature for each primer set (Table 2). The PCR conditions were as follows: initial denatur- MB-1 and MC-1, respectively, both of which are most likely ation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 0 s, 10 s representatives of a novel Desulfotomaculum subcluster, Ii annealing (temperatures are shown in Table 2), and extension at 72°C for 45 s, (Fig. 1). In addition, another phylotype, TJ-2, represented by 35 s, and 30 s for primer pairs DEM116f/DEM1164r and Ar109f/Ar912r and the two clones, was recovered from a thermophilic digested sludge other primers, respectively. Two tests were performed to confirm the specificity and was affiliated with subcluster Ib. No clones belonged to of the real-time PCR assays. First, melting-curve analysis was performed after each amplification step. Second, the sizes and the identities of PCR products lineages other than Desulfotomaculum cluster I. were confirmed by gel electrophoresis and subsequent clone library analysis. Abundance of Desulfotomaculum subcluster Ih bacteria. Expression analysis of dsrAB by RT-PCR. For reverse transcription (RT)-PCR Based on all known 16S rRNA gene sequences of subcluster Ih analysis, strain MGP was harvested under two different cultivation conditions, bacteria and clone sequences obtained in this study, a new 16S i.e., cells were incubated in the basal medium (20 mM propionate and 0.01% yeast extract) with and without sulfate (20 mM). Preparation of RNA from strain rRNA-targeted oligonucleotide probe, Ih820, was designed for MGP was done according to the method in a previous report (41) with slight FISH. First, the specificity of probe Ih820 was experimentally modification. The remaining DNA was digested with RNase-free DNase I (Pro- evaluated by using the perfectly matching target organism P. mega). The absence of contaminating genomic DNA in the RNA extract was thermopropionicum and the nontarget organism Moorella thermo- confirmed by PCR. RT-PCR was performed with a commercial RT-PCR kit acetica, which contains a single mismatch in the probe target (ReverTra Dash; TOYOBO) according to the manufacturer’s instructions. Two primer sets were used for amplification of dsrAB from strain MGP. MGP1F site. Nonspecific binding of probe Ih820 to M. thermoacetica (5Ј-ACGCACTGGAAACACG-3Ј) and MGP1R (5Ј-TATCAGCTATGTCGC cells could be avoided by adding equimolar amounts of unla- AGT-3Ј) amplify an approximately 480-bp-long fragment covering the 3Ј end of beled competitor probe to the hybridization solution and by dsrA, the intergenic region, and the 5Ј end of the dsrB gene. MGP9F (5Ј-CCA adjusting the formamide concentration to 20% (data not ACCCGTACTTCTTCT-3Ј) and MGP3R (5Ј-GTTAGCGCATCCGGATAC- 3Ј) amplify a 307-bp-long fragment of dsrB. Primers MGP1R and MGP3R were shown). Furthermore, probe DEM1164r (which was originally newly designed, while MGP1F and MGP9F are slightly modified versions of applied for PCR and membrane hybridization [52]) was opti- primers DSR1Fdeg and DSR9F, reported by Klein et al. (25) and Friedrich (14), mized with suitable reference cultures (P. thermopropionicum, VOL. 72, 2006 NON-SULFATE-REDUCING SYNTROPHIC DESULFOTOMACULUM CLUSTER I 2083 Downloaded from http://aem.asm.org/ on November 11, 2015 by University of Queensland Library

FIG. 1. Phylogenetic tree of Desulfotomaculum cluster I based on comparative analyses of 16S rRNA gene sequences and showing the phylogenetic positions of clones and strain MGP obtained in this study. Environmental clones obtained in this study are indicated in blue. Red indicates the isolated strain and clones from enrichment cultures established in this study. The initial tree was constructed with sequences greater than 1,000 nucleotides using the neighbor-joining method. Shorter sequences were subsequently inserted into the tree by using the parsimony insertion tool of ARB. The scale bar represents the estimated number of nucleotide changes per sequence position. The symbols at nodes show bootstrap values obtained after 1,000 resamplings. 2084 IMACHI ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 3. Relative abundances of bacteria belonging to copy numbers obtained by bacterial and archaeal primer sets) Desulfotomaculum cluster I and subcluster Ih as in the DNA extract. 16S rRNA genes of Desulfotomaculum determined by FISH cluster I bacteria were detected in all analyzed samples and Mean % of DAPI counts (SD) comprised 0.03% to 4.8% of the total prokaryotic 16S rRNA Desulfoto- gene pool. Subcluster Ih 16S rRNA genes could not be de- Desulfoto- maculum No. Environmental sample maculum tected in samples MB and MC but accounted for 0.03% to cluster I subcluster Ih (probe 3.9% of the total 16S rRNA genes in the other samples. (probe Ih820)

DEM1164r) Cultivation of Desulfotomaculum subcluster Ih bacteria. Downloaded from

1 TJ (thermophilic digested sludge) Ϫa Ϫ Taking advantage of known physiological properties of culti- 2 TO (thermophilic digested sludge) ϪϪvated subcluster Ih bacteria, such as P. thermopropionicum (6, 3 TR2 (thermophilic granular sludge) 1.1 (0.8) 0.5 (0.2) 4 TD (thermophilic granular sludge) 4.3 (1.2) 4.1 (1.1) 7, 16, 22, 23), we also aimed to enrich/isolate these micro- 5 MR1 (mesophilic granular sludge) Ͻ0.1 Ͻ0.1 organisms from the environmental samples. Primary anaerobic 6 MB (mesophilic granular sludge) Ͻ0.1 NDb 7 MC (mesophilic granular sludge) Ͻ0.1 ND enrichments were established at 37°C for the mesophilic- 8 MD (mesophilic digested sludge) ϪϪsludge samples and the paddy soil sample and at 55°C for the 9 RP (rice paddy soil) ϪϪthermophilic-sludge samples. Because almost all members of a Ϫ, FISH-based cell counting was impossible due to high autofluorescence of subcluster Ih are syntrophic anaerobes, 20% (vol/vol) of a http://aem.asm.org/ the sample and/or low signal of probe-stained bacteria. culture containing hydrogen- or formate-utilizing and aceti- b ND, not detected (below detection limit). clastic methanogens was added to all primary enrichment cultures; thermophilic cultures were inoculated with M. thermauto- ⌬ D. thermobenzoicum, D. thermosapovorans, and D. thermo- trophicus strain H, M. thermautorophicus strain type II, and M. cisternum) for the specific detection of Desulfotomaculum cluster thermophila, whereas mesophilic cultures were inoculated with I bacteria by FISH. Subsequently, the relative abundances cells of M. hungatei and M. concilii. In addition, because all of bacteria belonging to Desulfotomaculum cluster I and sub- known members of subcluster Ih can form spores, the inocula cluster Ih were determined for the nine environmental samples were pasteurized (80°C for 10 min) prior to the incubation. on November 11, 2015 by University of Queensland Library (Table 3). Subcluster Ih bacteria accounted for approximately Enrichment cultures were set up with a basal medium sup- 0.5% Ϯ 0.2% and 4.1% Ϯ 1.1% of the total DAPI (4Ј,6Ј- plemented with propionate (20 mM), ethanol (10 mM), and diamidino-2-phenylindole) counts in thermophilic-sludge sam- lactate (20 mM) because P. thermopropionicum can grow on ples TR2 and TD, respectively. The number of Ih820 probe- these substrates in syntrophic coculture with hydrogenotrophic positive cells in these samples closely corresponded to the methanogens. FISH analysis of subcluster Ih bacteria in the 27 number of DEM1164r probe-positive cells (Table 3), confirm- different enrichments showed that 10 enrichments were dom- ing that subcluster Ih bacteria were the dominant members of inated by probe Ih820-positive cells. The other 17 cultures the Desulfotomaculum clade in samples TR2 and TD. In con- contained various morphotypes of cells that could not be iden- trast, no Ih820-positive cells were detected in the mesophilic- tified as subcluster Ih bacteria. Five (2 propionate-degrading sludge samples MB and MC (Table 3). This observation is also enrichments from samples TR2 and TD, 2 ethanol-degrading consistent with the 16S rRNA gene library surveys, as all of the enrichments from samples TR2 and TD, and 1 lactate-degrad- MB and MC clones analyzed were affiliated with the novel ing enrichment from sample TR2) of the 10 cultures enriched subcluster Ii. in subcluster Ih were found to contain P. thermopropionicum Due to high autofluorescence or low probe-derived signals, as a dominant population (as revealed by FISH with probe microscopic quantification of the anaerobic digested-sludge TGP690) and were thus not used for further experiments. The samples TJ, TO, and MD and the rice paddy soil sample RP remaining five enrichments showed syntrophic propionate deg- was not possible. Hence, additional quantitative real-time PCR radation (Table 5) and contained Ih820-positive cells, which assays were performed (Table 4). The relative abundance of did not hybridize with the P. thermopropionicum probe. Micro- each target group was calculated as the percentage of the total scopic observation revealed that these cultures contained

16S rRNA gene copy number (deﬁned as the sum of the gene F420-autoﬂuorescent methanogen cells and spore-forming oval

TABLE 4. Relative abundances of Archaea, Bacteria, and Desulfotomaculum cluster I and subcluster Ih as determined by real-time PCR

Abundance (%)

No. Environmental sample Desulfotomaculum Desulfotomaculum Archaea Bacteria cluster I subcluster Ih 1 TJ (thermophilic digested sludge) 0.5 99.5 1.7 0.3 2 TO (thermophilic digested sludge) 1.2 98.8 0.3 0.1 3 TR2 (thermophilic granular sludge) 26.5 73.5 1.2 1.1 4 TD (thermophilic granular sludge) 7.3 92.7 4.8 3.9 5 MR1 (mesophilic granular sludge) 31.4 68.6 0.1 0.1 6 MB (mesophilic granular sludge) 5.0 95.0 0.03 NDa 7 MC (mesophilic granular sludge) 7.3 92.7 0.1 ND 8 MD (mesophilic digested sludge) 1.0 99.0 0.2 0.3 9 RP (rice paddy soil) 2.4 97.6 0.1 0.03

a ND, not detected (below detection limit). VOL. 72, 2006 NON-SULFATE-REDUCING SYNTROPHIC DESULFOTOMACULUM CLUSTER I 2085

TABLE 5. Stoichiometry of propionate conversion by microscopy and cultivation of the culture with a variety of Desulfotomaculum subcluster Ih bacteria a media containing substrates such as pyruvate, lactate, sucrose, enrichment cultures glucose, and yeast extract at 37°C or 55°C. Except for the Origin of Propionate Product formed Electron propionate-containing enrichment medium, none of these me- Enrichment Cultivation enrichment converted (mM) recovery name temp (°C) dia supported microbial growth. culture (mM) Acetate Methane (%) Partial characterization of strain MGP. Strain MGP was TJ JP 55 5.8 0b 10.1 99 cultivated in pure coculture with M. hungatei from an anaero- TO OP 55 8.2 0b 14.1 99 bic granular sludge treating an artiﬁcial wastewater (sample Downloaded from MR1 MGP 37 15.5 12.4 13.5 96 MD NP 37 7.3 0b 10.9 86 MR1) (Fig. 3). Cells of strain MGP were nonmotile and rod RP FP 37 9.9 10.0 7.4 98 shaped, with a length of 2.7 to 5.5 ␮m and a width of 1 ␮m (Fig.

a Products were measured after 80 days of incubation. 3). Spore formation was observed in late-log-phase cultures. b Acetate was most likely converted to methane, because Methanosaeta-like The speciﬁc growth rate on 20 mM propionate medium in cells were observed in the enrichment cultures under the microscope. coculture with M. hungatei was approximately 0.2 dayϪ1 (calculated based on methane production). 16S rRNA gene sequence analysis revealed that the strain belonged to Desulfo-

rods resembling Pelotomaculum species as dominant popula- tomaculum subcluster Ih (Fig. 1). The most closely related http://aem.asm.org/ tions. Growth and methane formation of the cultures occurred validly described organism of strain MGP was P. schinkii (95% after 40 to 90 days of incubation. Similar to cultures of other 16S rRNA sequence similarity with the P. schinkii rrnB 16S Pelotomaculum species (23, 43), it was difficult to maintain rRNA gene sequence) (6, 7). stable enrichments, i.e., growth and methane production some- Utilization of sulfate, sulfite, and organosulfonates by times stopped during the successive transfers of cultures. In Desulfotomaculum subcluster Ih bacteria. We evaluated the order to minimize unstable growth, cultures were not perma- sulfate- and sulfite-reducing abilities of subcluster Ih bacteria nently agitated (shaking of the vials for daily observation was that were highly enriched or isolated in this study, as well as done manually) for at least 40 days after starting the cultiva- those of other subcluster Ih bacteria, such as P. schinkii (6, 7)

tion. The five cultures continuously produced methane with and strains JT and JI (43; Qiu et al., unpublished). All incu- on November 11, 2015 by University of Queensland Library the concomitant degradation of propionate over six successive bation experiments were performed in triplicate. Cells of en- passages and contained probe Ih820-positive cells as the dom- richment cultures JP, OP, NP, and FP (after seven repeated inant bacterial population (Fig. 2). passages) and the pure (co)cultures were transferred into me- We performed 16S rRNA gene-based clone analyses to dium supplemented with sulfate (20 mM) or sulfite (1 and 2 characterize the dominant bacterial populations in these five mM), propionate (20 mM), and 2-bromoethane sulfonate (5 enrichments in more detail. Clone libraries were constructed mM) and incubated anaerobically at either 37°C or 55°C. How- by using a universal bacterial primer set, and 10 clones were ever, no growth and no depletion of electron donors were randomly selected and sequenced for each clone library. Most found after prolonged incubations (over 6 months) and when of the clones were affiliated with Desulfotomaculum subcluster dense cell suspensions were tested. In addition, sulfate and Ih (Fig. 1). Four out of five phylotypes retrieved from the sulfite reduction also never occurred in sulfate (20 mM) or enrichments (MGP, NP, JP, and OP) perfectly matched or sulfite (1 and 2 mM) medium that was supplemented with were very closely related to the phylotypes recovered from the propionate (20 mM) or terephthalate (1 mM) and external respective original samples. The only exception was phylotype electron carriers, such as menadione (500 ␮g/liter), 1,4-naph- ␮ ␮ FP from the rice paddy enrichment, which was clearly different thoquinone (500 g/liter), vitamin K1 (500 g/liter), or hemin from phylotypes RP-1 and RP-2 from the original rice paddy (50 ␮g/liter), and incubated for over 6 months (3, 19). soil sample (sequence similarities, 96.2 and 96.3%, respec- Moreover, we tested whether the growth of subcluster Ih tively). None of the sequences obtained was similar to Syntropho- bacteria was supported by organosulfonates, because some sul- bacter (2, 4, 17, 54) or Smithella (33) species, which are known fate reducers and closely related non-sulfate reducers can uti- mesophilic, syntrophic propionate-oxidizing Deltaproteobacteria. lize organosulfonates as electron acceptors for anaerobic res- Isolation of a novel syntrophic propionate-degrading strain. piration (29, 32). The pure (co)cultures P. thermopropionicum, We then attempted to isolate the subcluster Ih bacteria present P. schinkii, and strains JT, JI, and MGP and the enrichment in the enrichment cultures by employing a strategy successfully cultures JP, OP, NP, and FP (after eight successive transfers) applied for the isolation of P. thermopropionicum (23). Since were transferred into taurine (10 mM), isethionate (10 mM), we found that after seven successive transfers some contami- or cysteate (5 mM) medium supplemented with the respective nating microorganisms, which probably did not participate in electron donors (i.e., 10 mM propionate for P. thermopropion- propionate degradation, were still present in the enrichment icum, P. schinkii, strain MGP, and the highly enriched cultures cultures, we again pasteurized the cultures at 85°C for 30 min and 1 mM terephthalate for strains JT and JI). 2-Bromoethane or at 90°C for 20 min and serially diluted them into propionate sulfonate (5 mM) was added to the cultures to inhibit methan- medium containing hydrogenotrophic methanogens (M. therm- ogens, except for the P. thermopropionicum cultures. However, autotrophicus strain ⌬H and M. thermautotrophicus type II for growth and depletion of the electron donors were not observed thermophilic enrichments; M. hungatei for mesophilic enrich- after 3 months of incubation, although all experiments were ments). After a number of the serial dilution steps combined conducted in duplicate culture vessels. with pasteurization, a “pure” coculture with the hydrogenotro- dsrAB and apsA sequence analyses. To further investigate phic methanogen M. hungatei was obtained for enrichment whether subcluster Ih microorganisms harbor genes required MGP only at 37°C. The purity of this coculture was checked by for dissimilatory reduction of sulfate or sulfite, such as the 2086 IMACHI ET AL. APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/ on November 11, 2015 by University of Queensland Library VOL. 72, 2006 NON-SULFATE-REDUCING SYNTROPHIC DESULFOTOMACULUM CLUSTER I 2087

MGP cells were grown on propionate medium in the presence and absence of sulfate and subsequently subjected to RNA extraction. RT-PCR experiments using two newly designed primer pairs resulted in ampliﬁcation of PCR products of the expected size, indicating that both cultivation conditions al- lowed strain MGP to transcribe its dsrAB genes (Fig. 5). The sequences of PCR products obtained in this RT-PCR experi-

ment were completely identical to the dsrAB sequence of strain Downloaded from MGP that was obtained from DNA-based PCR ampliﬁcation, thereby proving the speciﬁcity of the RT-PCR assay.

DISCUSSION Distribution and abundance of Desulfotomaculum subcluster Ih bacteria. 16S rRNA gene sequences from members of Desulfotomaculum cluster I were detected in all methanogenic

FIG. 3. Phase-contrast micrograph of strain MGP grown on propi- http://aem.asm.org/ onate (20 mM) in coculture with M. hungatei. Bar, 10 ␮m. samples analyzed in this study (Fig. 1). These ﬁndings are in accordance with previous 16S rRNA gene surveys of granular sludges (48, 49), anaerobic petrol-contaminated sites (11), related Desulfotomaculum species, we screened for genes en- phthalate isomer-degrading consortia (43), toluene-degrading coding the dissimilatory (bi)sulﬁte reductase (DSR) and aden- consortia (13), and rice paddy soils (52, 57) and indicate that osine-5Ј-phosphosulfate reductase. We used various PCR Desulfotomaculum cluster I bacteria are ubiquitously distrib- primers targeting conserved sites in the genes for the alpha and uted in methanogenic environments. The relative natural abun- beta subunits of DSR (dsrAB) and the gene for the alpha dance of this group varied from approximately 0.05 to 7% of

subunit of adenosine-5Ј-phosphosulfate reductase (apsA) (9, the total bacterial or microbial populations, as determined on November 11, 2015 by University of Queensland Library 14, 25). A number of PCR experiments were performed under previously by different molecular methods (10, 21, 51, 52). In different reaction conditions (i.e., different Mg2ϩ concentra- comparison, our quantitative PCR data for Desulfotomaculum tions in the PCR buffer, annealing temperatures from 45°C to cluster I bacteria were also in the same range (0.03 to 4.8% of 59°C, or reamplification of the initial PCR products) for the the total microbial 16S rRNA gene pool). Additionally, we pure (co)cultures P. thermopropionicum, P. schinkii, and strains showed that subcluster Ih-type cells represent the majority of JT, JI, and MGP and the enrichment cultures JP, OP, NP, and the Desulfotomaculum cluster I cells in most of our samples. FP (after eight successive transfers). However, only strain These observations are also consistent with previous reports MGP and the thermophilic enrichment culture JP yielded a (21, 51, 52). For example, quantitative dot blot and real-time PCR product for dsrAB (1,797 bp) and apsA (377 bp), respec- PCR analyses of an Italian rice soil 16S rRNA gene clone tively. Sequencing and comparative analysis of both PCR prod- cluster, which also belongs to subcluster Ih (Fig. 1) (52), dem- ucts demonstrated that the sequences were homologous to the onstrated that its population size was almost equivalent to the respective target genes of Desulfotomaculum species. In more size of the total Desulfotomaculum cluster I community (51, detail, the dsrAB sequence from strain MGP formed a new 52). At first glance, the proposed numerical dominance of lineage in the DsrAB tree and was related to sequences from subcluster Ih among environmental Desulfotomaculum cluster other gram-positive bacteria (including the Desulfotomaculum I members is inconsistent with the findings of Hristova et al. subcluster Ia and If bacteria) (Fig. 4). The apsA sequence from (21). They reported, based on quantitative dot blot hybridiza- the thermophilic enrichment culture JP formed a monophy- tion with the 16S rRNA-targeted probe S-*-Dtm(bcd)-0230-a- letic lineage with the subcluster Ib Desulfotomaculum species A-18, that members of the subclusters Ib, Ic, and Id were the Desulfotomaculum sapomandens and Desulfotomaculum geo- most abundant Desulfotomaculum cluster I bacteria in a variety thermicum (deduced amino acid sequence similarity, 81.6%) of anoxic environments. However, reevaluation of the speci- in all trees (data not shown). However, it remains unclear ficity of the probe showed that it also targets the majority of whether this apsA sequence originated from a subcluster Ih 16S rRNA gene sequences affiliated with subcluster Ih, sug- bacterium, because (i) the amplified apsA fragment was too gesting that the results of Hristova et al. (21) and of our study short for a reliable phylogenetic analysis and (ii) the enrich- are not necessarily contradictory. In summary, our findings ment culture JP still contained some contaminating micro- show that Desulfotomaculum subcluster Ih constitutes an im- organisms. portant fraction of the microbial community in a wide variety dsrAB expression in strain MGP. We performed RT-PCR of methanogenic environments, highlighting its ecological im- experiments by using RNA extracts from strain MGP to check pact in anoxic systems, usually with a low concentration of whether its dsrAB genes are transcribed. For this purpose, sulfate. However, the ubiquity of subcluster Ih bacteria will

FIG. 2. In situ hybridization of novel subcluster Ih bacteria in syntrophic propionate-degrading enrichment cultures with Cy3-labeled Ih820 probe. Phase-contrast micrographs (A, C, E, G, and I) and ﬂuorescence micrographs (B, D, F, H, and J) of the same ﬁelds are shown. (A and B) JP enrichment culture inoculated from environmental sample TJ; (C and D) OP enrichment culture from environmental sample TO; (E and F) MGP enrichment culture from environmental sample MR1; (G and H) NP enrichment culture from environmental sample MD; (I and J) FP enrichment culture from environmental sample RP. Bars, 10 ␮m. 2088 IMACHI ET AL. APPL.ENVIRON.MICROBIOL. Downloaded from http://aem.asm.org/ on November 11, 2015 by University of Queensland Library

FIG. 4. Phylogenetic consensus tree (based on FITCH distance matrix analysis) of DsrAB amino acid sequences deduced from dsrAB sequences of greater than 1,750 bases showing the afﬁliation of Pelotomaculum species strain MGP (indicated in boldface type). The bar indicates 10% estimated sequence divergence. Polytomic nodes connect branches for which a relative order could not be determined unambiguously by applying distance matrix, maximum-parsimony, and maximum-likelihood tree-building methods. Parsimony bootstrap values (100 resamplings) for branches are indicated by solid (Ͼ90%) or open (75 to 90%) circles. Branches without circles had bootstrap values of less than 75%. Gray boxes indicate different subclusters of Desulfotomaculum cluster I. LA-dsrAB Firmicutes is a low-GϩC-content gram-positive bacterium with laterally acquired dsrAB genes.

need to be further surveyed for other methanogenic environ- tomaculum cluster I are not always indicative of the presence of ments, since our investigation was confined to eight anaerobic sulfate reducers and must be carefully interpreted. sludges and one rice paddy soil. Another interesting physiological feature of subcluster Ih Ecophysiology of Desulfotomaculum subcluster Ih bacteria. bacteria is their ability to catabolize organic substances in Their close phylogenetic relationship with classical Desulfo- syntrophic associations with hydrogenotrophic methanogens. tomaculum species suggests that members of Desulfotomaculum With the exception of Cryptanaerobacter phenolicus (24), all subcluster Ih are sulfate-reducing bacteria. However, all subclus- previously isolated strains and cultures of subcluster Ih ob- ter Ih-containing cultures (including P. thermopropionicum, P. tained in this study showed this unique phenotype, although schinkii, and strains JT and JI [42a, 43]) investigated in this study the range of degradable substrates differed among strains/cul- exhibited no growth with sulfate, sulfite, or organosulfonates, tures. The physiology of the phenol degrader C. phenolicus corroborating and extending previous findings on the physiolog- (previously known as strain 7) has not been fully examined, and ical characteristics of this group (7, 22, 24). Consequently, envi- thus, it is currently unknown whether this strain can perform ronmentally retrieved 16S rRNA genes belonging to Desulfo- syntrophic substrate oxidation in cooperation with hydro- VOL. 72, 2006 NON-SULFATE-REDUCING SYNTROPHIC DESULFOTOMACULUM CLUSTER I 2089

Presence and expression of dsrAB in strain MGP. Although various PCR conditions were used, a dsrAB sequence was recovered only from the coculture containing strain MGP. The other Desulfotomaculum subcluster Ih bacteria either do not possess dsrAB or their dsrAB genes contained strong mis- matches in the primer target sites, preventing ampliﬁcation (58). Rapid accumulation of mutations in dsrAB would be

particularly likely in these strains, as they apparently no longer Downloaded from use their dissimilatory (bi)sulfite reductase. Additional exten- sive experiments (for example, heterologous Southern hybridization analyses using dsrAB-targeted polynucleotide probes), FIG. 5. RT-PCR amplification of dsrAB mRNA and 16S rRNA from strain MGP. Lane M, marker; lanes 1, 4, and 7, DNA of strain which were beyond the scope of this study, will be required to MGP (positive controls); lanes 2, 5, and 8, RNA extracted from strain clarify whether the genomes of these strains still carry dsrAB MGP grown on only propionate (20 mM); lanes 3, 6, and 9, RNA (pseudo)genes. extracted from strain MGP grown on propionate (20 mM) plus sulfate The dsrAB genes from strain MGP, representing the first (20 mM). PCR products were amplified by the following primer sets:

MGP1F/MGP1R (lanes 1, 2, and 3); MGP9F/MGP3R (lanes 4, 5 and entry of a Desulfotomaculum cluster Ih member in the dsrAB http://aem.asm.org/ 6); and 341f/907r (lanes 7, 8, and 9). database, is affiliated with Desulfotomaculum species but formed a new lineage within the group (Fig. 4). This is in accordance with the 16S rRNA-based phylogeny of strain MGP. Previous comparative analyses of 16S rRNA- and genotrophic microbes (24, 31). Given their recognized pheno- DsrAB-based trees have shown that multiple lateral transfers types and wide occurrence in low-sulfate, methanogenic envi- of dsrAB have occurred among sulfate- or sulfite-reducing pro- ronments, descendants of the Desulfotomaculum subcluster Ih karyotes (25, 58). For example, members of Desulfotomaculum branch most likely function as non-sulfate-reducing, syntrophic cluster I are phylogenetically separated in the DsrAB tree. degraders of organic substrates in situ. This hypothesis re- Desulfotomaculum subclusters Ib to Ie form a monophyletic on November 11, 2015 by University of Queensland Library ceived further independent support from a recent study, which lineage with the deltaproteobacterial species Desulfobacterium showed, by using rRNA-based stable-isotope probing, that anilini and have received their dsrAB genes laterally from a Pelotomaculum species were actively involved in syntrophic deltaproteobacterial ancestor (25, 58). In contrast, the dsrAB propionate oxidation in rice paddy soil (37). genes of strain MGP are affiliated with Desulfotomaculum sub- Obligate syntrophic lifestyle of strain MGP? Similar to pre- clusters Ia and If, suggesting that the strain acquired its dsrAB vious cultivation attempts (22, 23, 43), the growth of subcluster genes by vertical transmission from a gram-positive ancestor Ih bacteria cultivated in this study was very slow and unstable. (Fig. 4). An interesting feature associated with the dsrAB genes To enhance the stability of the growth of syntrophic, substrate- of strain MGP is that these genes are transcribed in the pres- degrading cultures, a considerable amount (20% [vol/vol] in- ence and absence of sulfate (Fig. 5), although the MGP-con- oculum) of pure cultures of hydrogen- or formate-utilizing taining coculture could not reduce sulfate, sulfite, or organo- methanogens and aceticlastic methanogens was added to all sulfonates, which are typical (precursors of) substrates of primary enrichment cultures. Unexpectedly, growth stability dissimilatory (bi)sulfite reductases (29, 30, 32). The recovered did not improve, and thus, only one propionate-degrading dsrAB sequence fragment of strain MGP does not contain strain, MGP, was finally obtained in a binary mixed pure cul- unusual errors (such as unexpected stop codons or frame shifts ture from an anaerobic granular sludge treating artificial within the genes) indicative of pseudogenes. In addition, both wastewater. In contrast to many other propionate-oxidizing subunits in the DsrAB amino acid sequence of strain MGP syntrophs, which are also able to respire sulfate (2, 4, 17, 42, contain the conserved cysteine motif consensus sequences Cys-

54) or dismutate crotonate in pure culture (33), this strain grew X5-Cys and Cys-X3-Cys that are essential for binding the only in syntrophic association with hydrogenotrophic methano- [Fe4S4]-siroheme cofactor of sulfite reductases (5, 18). While gens. Addition of various substrates did not support axenic these findings suggest that these dsrAB genes might still encode growth of the strain. Similarly, the mesophile P. schinkii can a functional protein, their potential roles in the physiology of also grow only on propionate in coculture with hydrogenotro- strain MGP remains unresolved. phic methanogens and has thus been reported as the “first true The apparent discrepancy between the presence of dsrAB obligately syntrophic propionate-oxidizing bacterium” (6, 7). and the inability to reduce sulfate/sulfite of strain MGP might The close phylogenetic relationship to P. schinkii and the cur- reflect a relatively recent evolutionary transition. According to rently known physiological capabilities suggest that strain the 16S rRNAgene-based tree, subcluster Ih bacteria have only MGP is also an obligate syntroph. Although we have recently recently diverged from members of the other subclusters (Fig. isolated an additional strain, JI, which grows only on aro- 1), indicating a common endospore-forming, sulfate-reducing matic compounds in coculture with hydrogenotrophic methan- ancestor for all bacteria of the Desulfotomaculum cluster I ogens and belongs to Desulfotomaculum subcluster Ih (Qiu branch. In order to cope with the usually limited availability of et al. unpublished), obligate syntrophy is not a phenotype sulfate in methanogenic ecosystems, subcluster Ih bacteria common to all members of this lineage. For example, C. could have specialized in living syntrophically with methano- phenolicus degrades phenol to benzoate (24), and P. thermo- genic archaea. Consequently, the capability for dissimilatory propionicum ferments pyruvate and fumarate (22, 23) in pure sulfate/sulfite reduction might have been lost in the course of culture. this evolutionary process. In this respect, the presence of tran- 2090 IMACHI ET AL. APPL.ENVIRON.MICROBIOL. scribed dsrAB genes might represent a genetic and/or gene- 14. Friedrich, M. W. 2002. Phylogenetic analysis reveals multiple lateral trans- Ј regulatory leftover in strain MGP. Inactivation and/or deletion fers of adenosine-5 -phosphosulfate reductase genes among sulfate-reducing microorganisms. J. Bacteriol. 184:278–289. of genes other than dsrAB, such as dsrMKJOP (hmeABCDE) 15. Großkopf, R., P. H. Janssen, and W. Liesack. 1998. Diversity and structure and qmoABC, which might be essential for electron transfer of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl. during sulfate respiration (39), could be responsible for the Environ. Microbiol. 64:960–969. apparent lack of this phenotype in subcluster Ih bacteria. 16. Harmsen, H. J. M. 1996. Detection, phylogeny and population dynamics of Similar evolutionary forces could have shaped Sporotomacu- syntrophic propionate-oxidizing bacteria in anaerobic granular sludge. Ph.D. thesis. Wageningen Agricultural University, Wageningen, The Netherlands. lum species (3, 44), which are also not able to respire with 17. Harmsen, H. J. M., B. L. M. Van Kuijk, C. M. Plugge, A. D. L. Akkermans, Downloaded from sulfate or sulfite, although they belong to Desulfotomaculum W. M. de Vos, and A. J. M. Stams. 1998. Syntrophobacter fumaroxidans sp. subcluster Ib and possess dsrAB genes (Fig. 1 and 4). It is ex- nov., a syntrophic propionate-degrading sulfate-reducing bacterium. Int. J. Syst. Bacteriol. 48:1383–1387. pected that comparative analysis of genome sequences of closely 18. Hipp, W. M., A. S. Pott, N. Thum-Schmitz, I. Faath, C. Dahl, and H. G. related sulfate-reducing and non-sulfate-reducing microorgan- Tru¨per. 1997. Towards the phylogeny of APS reductase and sirohaem sulfite reductases in sulfate-reducing and sulfur-oxidizing prokaryotes. Micro- isms will yield important insights into recent evolutionary adap- biology 143:2891–2902. tations of gram-positive sulfate reducers and the mechanisms 19. Hippe, H., A. Hagenauer, and R. M. Kroppenstedt. 1997. Menadione re- underlying the apparent loss of their ancestral phenotype. quirement for sulfate-reduction in Desulfotomaculum aeronauticum, and emended species description. Syst. Appl. Microbiol. 20:554–558.

20. Hiraishi, A. 1992. Direct automated sequencing of 16S rDNA amplified by http://aem.asm.org/ ACKNOWLEDGMENTS polymerase chain reaction from bacterial cultures without DNA purification. 15: We thank Frank de Bok, Sanae Sakai, Masashi Hatamoto, Yasushi Lett. Appl. Microbiol. 210–213. 21. Hristova, K. R., M. Mau, D. Zheng, R. I. Aminov, R. I. Mackie, H. R. Hirukoi, Akinobu Nakamura, Kucivilize Pairaya, Kanao Otake, Satoshi Gaskins, and L. Raskin. 2000. Desulfotomaculum genus- and group-specific Hanada, Akira Hiraishi, Eiji Masai, Hirofumi Hara, Kazuaki Syutsubo, small-subunit rRNA hybridization probes for environmental studies. Envi- and Tadashi Tagawa for their help. ron. Microbiol. 2:143–159. This study was financially supported by the New Energy and Indus- 22. Imachi, H., Y. Sekiguchi, Y. Kamagata, S. Hanada, A. Ohashi, and H. trial Technology Development Organization (NEDO) and the Japan Harada. 2002. Pelotomaculum thermopropionicum gen. nov. sp. nov., an Society for the Promotion of Science (JSPS), Tokyo, Japan. A.L. and anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium. Int. J. M.W. were supported by the European Community (Marie Curie Syst. Evol. Microbiol. 52:1729–1735. Intra-European Fellowship within the 6th Framework Programme), 23. Imachi, H., Y. Sekiguchi, Y. Kamagata, A. Ohashi, and H. Harada. 2000. Cultivation and in situ detection of a thermophilic bacterium capable of on November 11, 2015 by University of Queensland Library the Fonds zur Fo¨rderung der wissenschaftlichen Forschung, and the ϩ oxidizing propionate in syntrophic association with hydrogenotrophic methan- bmb f (project 01 LC 0021A-TP2 in the framework of the BIOLOG ogens in a thermophilic methanogenic granular sludge. Appl. Environ. Mi- II program). crobiol. 66:3608–3615. 24. Juteau, P., V. Coˆte´, M.-F. Duckett, R. Beaudet, F. Le´pine, R. Villemur, and REFERENCES J.-G. Bisaillon. 2005. 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