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1 Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
2 Department of Medical Microbiology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
Correspondence
Frank de Bok
Frank.deBok{at}wur.nl
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Published online ahead of print on 29 April 2005 as DOI 10.1099/ijs.0.02880-0.
The GenBank/EMBL/DDBJ accession numbers for the ‘spore A’ and ‘spore B’ 16S rRNA gene sequences of strain HHT are respectively X91169 and X91170.
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). Propionate oxidation is an important step in this process, which requires obligately syntrophic consortia of acetogenic bacteria and methanogenic archaea. The methanogens make propionate oxidation energetically feasible by keeping the concentrations of the products H2 and formate extremely low (de Bok et al., 2004; Schink, 1997; Schink & Stams, 2002). Most of the syntrophic propionate-oxidizing bacteria isolated so far belong to the Syntrophobacter cluster within the ‘Deltaproteobacteria’. Syntrophobacter species are able to use sulfate as the electron acceptor for propionate oxidation (Harmsen et al., 1993; Wallrabenstein et al., 1995). In addition, they can grow by fermentation of pyruvate and fumarate. Smithella propionica LYPT, which was isolated more recently, is phylogenetically related to the genus Syntrophus (Liu et al., 1999). This Gram-negative propionate-oxidizer lacks the ability to reduce sulfate and uses a different pathway to oxidize propionate from that used by Syntrophobacter strains. However, Smithella propionica LYPT is able to grow axenically on crotonate (de Bok et al., 2001; Liu et al., 1999). Syntrophic propionate oxidation is not restricted to Gram-negative bacteria. Wu et al. (1992) described a Gram-positive, syntrophic propionate-oxidizing bacterium that is able to produce spores. Apart from its grouping within the Gram-positive bacteria, no further physiological characterization was reported. Two thermophilic, Gram-positive, syntrophic propionate-oxidizing bacteria have been described, Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum TPOT, which grows axenically on several substrates and, like Syntrophobacter strains, can use sulfate as an electron acceptor (Plugge et al., 2002), and Pelotomaculum thermopropionicum SIT, which lacks the ability to reduce sulfate but ferments pyruvate and fumarate in pure culture (Imachi et al., 2000, 2002). In this study, we describe a mesophilic, spore-forming, syntrophic propionate-oxidizing bacterium, strain HHT, which is also closely related to Pelotomaculum thermopropionicum SIT and the genus Desulfotomaculum. This strain was obtained as a defined co-culture with Methanospirillum hungatei JF-1T and could not grow axenically on any of the substrates tested. Methanospirillum hungatei JF-1T (=DSM 864T), Methanobacterium formicicum MFNT (=DSM 1535NT) and Methanobrevibacter arboriphilus AZ (=DSM 744) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). A bicarbonate-buffered mineral medium was used, with the following composition: 3 mM Na2HPO4, 3 mM KH2PO4, 5·6 mM NH4Cl, 0·75 mM CaCl2, 0·5 mM MgCl2, 5 mM NaCl, 50 mM NaHCO3, 1 mM Na2S, 7·5 µM FeCl2, 1 µM H3BO3, 0·5 µM ZnCl2, 0·1 µM CuCl2, 0·5 µM MnCl2, 0·5 µM CoCl2, 0·1 µM NiCl2, 0·1 µM Na2SeO3, 0·1 µM Na2WO4, 0·1 µM Na2MoO4, 0·5 mg EDTA l–1 and the following vitamins (mg l–1): 0·02 biotin, 0·2 nicotinic acid, 0·5 pyridoxine, 0·1 riboflavin, 0·2 thiamin, 0·1 cyanocobalamin, 0·1 p-aminobenzoic acid, 0·1 pantothenic acid, 0·1 lipoic acid and 0·1 folic acid. Methanogenic archaea were cultured routinely at 37 °C in 120 ml serum flasks with 50 ml medium and a gas phase of 1·7 atm. H2/CO2 (80 : 20, v/v). Prior to inoculation of the propionate-oxidizing bacteria, CH4 and residual H2 were removed and replaced by 1·7 atm. N2/CO2 (80 : 20, v/v). Propionate-oxidizing bacteria were inoculated in H2/CO2-pregrown cultures of Methanospirillum hungatei JF-1T, Methanobacterium formicicum MFNT or Methanobrevibacter arboriphilus AZ. Routinely, 5 % of a pasteurized (20 min, 85 °C) methanogenic co-culture was used as the inoculum. After addition of sodium propionate (20 mM final concentration), the cultures were incubated at 37 °C. Percoll density-gradient centrifugation was carried out under anoxic conditions using a glove box with N2/H2 (96 : 4, v/v) as the gas phase and airtight centrifuge tubes for centrifugation steps outside the glove box. Traces of oxygen were removed from the glove box by circulating the gas phase over a platinum catalyst column. Cells were collected from a 0·5 l propionate-oxidizing enrichment culture by centrifugation at 16 000 g. The cell pellet was resuspended in a mixture of 70 % Percoll and 50 mM sodium phosphate, pH 7·5, containing 2 mM Na2S. The cells were separated in a Percoll gradient, which was generated in a 9 ml centrifuge tube at 30 000 g and 18 °C for 30 min.
Organic acids were measured with a Spectrasystem HPLC system equipped with an autosampler and refractometer. The acids were separated on a Polyspher OAHY column (30 cmx6·5 mm; Merck) in 0·005 M H2SO4 at a flow rate of 0·6 ml min–1 and a column temperature of 60 °C. The acids eluting from the column were quantified by differential refractometry. H2 and CH4 were measured using a Packard-Becker 417 gas chromatograph equipped with a thermal conductivity detector and a molecular sieve 13X (60/80 mesh). The column temperature was 50 °C and the carrier gas was argon at a flow rate of 30 ml min–1. To determine whether strain HHT uses a randomizing pathway of propionate oxidation, a co-culture was grown in the presence of 5 mM 3-13C-propionate (de Bok et al., 2001). At the end of the exponential growth phase, a sample was withdrawn from this culture and analysed for isotopes using 13C-NMR spectroscopy. A proton-decoupled 13C-NMR spectrum was recorded at 75·47 MHz on a Bruker AMX-300 NMR spectrometer as described previously (de Bok et al., 2001).
Nucleic acids were isolated from 10 ml of a late-exponential-phase enrichment culture as described previously (Harmsen et al., 1995). Bacterial 16S rRNA genes present in the enrichment culture were amplified by PCR as described by Harmsen et al. (1993) using a set of universal 16S rRNA-based primers containing restriction sites (in bold) for BamHI and PstI to facilitate cloning: the forward primer (5'-CACGGATCCGGACGGGTGAGTAACACG) corresponded to Escherichia coli positions 106–124 and the reverse primer (5'-GTGCTGCAGGGTTACCTTGTTAC GACT) to E. coli positions 1493–1510. Recombinant plasmids were obtained as described previously using pUC18 as cloning vector and E. coli TG1 as host (Harmsen et al., 1996). DNA sequencing was done using the dideoxy chain-termination method (Sanger et al., 1977) adapted for cycle-sequencing with Taq polymerase as described in the Life Science Technologies (now Invitrogen) manual. All enzymes for DNA manipulations were obtained from Life Science Technologies. Unlabelled oligonucleotides were purchased from Pharmacia. Partial 16S rRNA gene nucleotide sequences were aligned with those of other bacteria, taking into account sequence similarity and higher order structure, using the alignment tool of the ARB software package (Strunk & Ludwig, 1995). Slot-blot hybridization experiments were performed on Hybond N+ filters (GE Healthcare). Nucleic acid samples containing approximately 50 ng DNA were applied to the membrane with a Hybridot manifold (Life Science Technologies) and immobilized according to Church & Gilbert (1984). The membranes were pretreated with hybridization buffer (0·5 M sodium phosphate, pH 7·2, 7 % SDS, 1 % BSA and 1 mM EDTA) for 30 min prior to hybridization with 100 ng [
-32P]ATP-labelled probes. All membranes were hybridized at 40 °C and washed in 1 % SDS, 1x SSC (0·15 M NaCl, 0·015 M sodium citrate, pH 7·0) wash buffer at 50 °C. The membranes were exposed to a Kodak X-ray film or exposed to a phosphorimager screen. The screen was then scanned for radioactive response on a PhosphorImager (Molecular Dynamics). Digital signals were processed by the manufacturer's software (ImageQuant). Cells were prepared for fluorescent in situ hybridization (FISH) either by mixing culture samples 1 : 1 directly with 8 % paraformaldehyde or by concentrating the culture samples by centrifugation and then resuspending the cell pellets in PBS (130 mM NaCl, 10 mM sodium phosphate, pH 7·2) containing 4 % paraformaldehyde. Cells were fixed for 2 h and then washed with PBS and resuspended in PBS/ethanol (1 : 1). Alternatively, cells were fixed by mixing the culture samples 1 : 1 with ice-cold 96 % ethanol or resuspending cell pellets in PBS/ethanol (1 : 1). Samples were stored at –20 °C. Samples were dried onto glass slides (10 µl per well), washed with Milli-Q water and then dehydrated in an increasing ethanol series (3 min each in 50, 80 and 96 % v/v ethanol). To test whether lysozyme improved the permeabilization, slides were incubated in a lysozyme solution (100 mg ml–1) for 10 min at 50 °C. The cells were hybridized for 1·5 h with fluorescein- or Cy3-labelled oligonucleotide probes at 50 °C and formamide at 0, 10, 20, 30, 40 and 50 %, according to Amann (1996). Fluorescent cells were detected with an Olympus BH2 epifluorescence microscope.
The propionate-oxidizing culture was enriched from freeze-dried granular sludge from a full-scale upflow anaerobic sludge bed (UASB) reactor treating sugar beet waste (CSM, Breda, The Netherlands). After freeze-drying, the sludge had been stored aerobically for more than 2 years at room temperature. Bottles with freshly prepared medium (50 ml) were inoculated with 0·2 g freeze-dried granular sludge. After addition of 20 mM sodium propionate and 10 % (v/v) of a H2/CO2-grown culture of Methanospirillum hungatei JF-1T, the enrichment culture was incubated at 37 °C. After 3 months, the propionate was depleted and CH4 was produced. Acetate was detected in only small amounts, indicating the presence of acetoclastic methanogens. Propionate-oxidizing bacteria were further purified by repeated rounds of pasteurization for 30 min at 85 °C and subculturing in the presence of Methanospirillum hungatei JF-1T. The highest dilution with growth (usually dilution 108) was always used for subculturing. After six transfers, the culture consisted of two morphotypes, Methanospirillum hungatei JF-1T and a rod-shaped bacterium, which formed endospores (Fig. 1). No other bacteria were observed microscopically. However, upon addition of glucose, a long-rod-shaped bacterium developed. This bacterium remained, even after additional rounds of pasteurization and subculturing. The doubling time of the culture was approximately 7 days and the culture had a lag phase varying from 1 to 8 weeks.
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). The propionate-oxidizing bacteria were 1x2·0–2·5 µm in size; non-motile and produced centrally located endospores in the late exponential phase. Sequence analysis of the 16S rRNA genes amplified from this culture revealed that both sequences obtained from the enrichment culture were still present in equal amounts in this culture.
An enrichment culture that had been pasteurized and subcultured at least 15 times under syntrophic conditions was used for phylogenetic analysis. PCR amplification of the 16S rRNA genes from nucleic acids isolated from this culture resulted in a product of the expected size of 1·4 kb. This fragment was digested with BamHI and PstI and ligated in pUC18 linearized with the same enzymes. The ligation products were transformed into E. coli TG1 cells, which resulted in 38 recombinant plasmids. Ten of the recombinant plasmids were characterized by sequence analysis of the insert DNA using the universal 16S rRNA primer 1115 (E. coli positions 1100–1115; Lane, 1991). Four of these plasmids contained inserts with identical sequences of the V6 region of the 16S rRNA, indicated as ‘spore A’ sequences. Five other plasmids also contained identical sequences, indicated as ‘spore B’. One plasmid contained an unidentified sequence. Two plasmids containing a spore A or B 16S rRNA gene sequence were selected for further analysis and their inserts were sequenced completely, resulting in sequences of 1361 and 1362 bp, respectively. These sequences have been deposited in the EMBL database with accession numbers X91169 (spore A) and X91170 (spore B). The similarity between the two 16S rRNA gene sequences was 96·8 %. Specific oligonucleotide probes were designed against the V6 region of the type A and type B 16S rRNA gene sequences (5'-GGACTACTGACACCTTTGTGTCTC and 5'-GAGACTGTCGGATACTTTCATCC for A and B, respectively). A slot blot containing approximately 100 ng of each of the 38 plasmids was hybridized with both probes to determine the ratio in which the clone library represented the two sequence types. In addition, a comparable amount of DNA obtained from five different PCR amplifications was added to the blot to determine whether selective cloning had occurred. Thirteen of the 38 plasmids gave a positive signal with the type A-specific probe and 17 plasmids gave a positive signal with the type B-specific probe. All five PCR products hybridized with both probes with approximately the same efficiency, indicating that the two 16S rRNA gene sequences were present in equal amounts after amplification.
Sequence heterogeneities in the rRNA operons are not an unusual property of bacteria, as this has also been described for other Gram-positive bacteria such as Paenibacillus polymyxa (Nubel et al., 1996), Bifidobacterium adolescentis (Satokari et al., 2001) and Clostridium perfringens (Shimizu et al., 2001). However, to exclude the possibility that there were two closely related bacterial species left in the co-culture, FISH was used to target both sequences in individual cells. Based on sequence differences observed in the V2 regions of the 16S rRNA gene sequences (Table 1), two fluorescently labelled oligonucleotide probes were designed to determine whether the type A and type B sequences belonged to a single organism. The best hybridization results were obtained with early exponential-phase cells that were fixed with paraformaldehyde and hybridized in the presence of 10 % formamide. Both V2 probes were detected in the same cells and none of the cells hybridized with only one of these probes, although faint signals were detected in some cells (Fig. 1). To exclude cross-hybridization with non-target bacteria, three environmental strains, Succiniclasticum ruminis DSM 9236T, Sporomusa paucivorans DSM 3697T and Clostridium sporosphaeroides DSM 1294T, and five clinical strains (identified as Eubacterium cylindroides, Clostridium butyricum, Clostridium nexile, Fusobacterium mortiferum and Clostridium cadaveris) were fixed and hybridized with both V2 probes under the conditions which were optimal for strain HHT, but a signal was detected for none of these strains. Based on these results, we conclude that strain HHT contains two different 16S rRNA gene sequences which are both expressed during syntrophic growth on propionate.
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HT and Z245. The syntrophic culture converted 1 mol propionate to about 0·9 mol acetate and 0·7 mol CH4. The growth rate of strain HHT depended strongly on the number of methanogens present. During enrichment, only 10 % (v/v) of a H2/CO2-grown culture of Methanospirillum hungatei JF-1T was added, and the maximum doubling time observed in these cultures was only approximately 7 days. For further isolation procedures, the culture was inoculated into batches in which Methanospirillum hungatei JF-1T was pregrown on H2/CO2. In these batches, the doubling time was close to 3 days, most likely as a result of the high density of Methanospirillum hungatei JF-1T. The time needed for a 109-diluted culture to show visible growth suggests that the doubling time is even shorter under optimal conditions, approximately 1·5 days. Yeast extract (1 g l–1) and fumarate stimulated growth, but the culture was not able to grow on these compounds in the absence of propionate and Methanospirillum hungatei JF-1T. When fumarate (20 mM) was added, propionate was degraded more rapidly and, in addition to acetate, malate and succinate were produced. Addition of fumarate did not affect the biomass yield, which was determined by measuring the optical density. Other substrates which were tested for syntrophic growth (at 20 mM unless indicated), but which were not utilized, included lactate, pyruvate, fumarate, malate, succinate, acetate, citrate, 
-ketoglutarate, butyrate, 3-hydroxybutyrate, 4-hydroxybutyrate, isobutyrate, crotonate, benzoate, glucose, fructose, xylose, methanol, ethanol, isopropanol, propanol, glycerol, butanediol, glycine, aspartate, serine, alanine, glutamate and proline (5 mM). The electron acceptors sulfate, thiosulfate, sulfite, nitrate, chlorate, iron(III) EDTA, fumarate, proline, glycine, crotonate, anthraquinone disulfonate (2·5 mM) and elemental sulfur (4 g l–1) did not support axenic growth of pasteurized cultures in the presence of propionate. Pasteurized cultures did not grow on H2/CO2, formate or pyruvate.
Most syntrophic propionate-oxidizing bacteria use the randomizing methylmalonyl CoA pathway to oxidize propionate (Houwen et al., 1990, 1991; Plugge et al., 1993). An alternative pathway, which has recently been proposed for Smithella propionica LYPT, explains the presence of a non-randomizing pathway in methanogenic ecosystems and enrichment cultures (de Bok et al., 2001). A 13C-NMR experiment with strain HHT grown on 3-13C-propionate yielded both 1- and 2-13C-acetate, indicating that the organism uses the methylmalonyl CoA pathway (data not shown). The presence of this pathway has also been demonstrated in the closely related Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum TPOT (Plugge et al., 2002) by measuring some of the key enzymes of the pathway. We tested strain HHT for axenic growth on several intermediates of this pathway, including several combinations with propionate, but growth was not observed in any of these cultures. As indicated above, fumarate stimulated syntrophic growth on propionate in this organism, probably by preventing the energetically unfavourable oxidation of succinate to fumarate during propionate oxidation. Syntrophobacter species are able to ferment intermediates due to their ability to couple the energetically unfavourable oxidations to fumarate reduction. Apparently, strain HHT is not able to couple the electrons released in the three oxidation steps during propionate oxidation to fumarate reduction. Thus, strain HHT seems to represent the first true obligately syntrophic anaerobic bacterium.
Comparative sequence analysis of the 16S rRNA gene sequences derived from the enrichment culture (spore A and spore B) with other sequences revealed that strain HHT clusters phylogenetically with species of the genus Desulfotomaculum and is most closely related to Pelotomaculum strain FP, an organism for which two sequences (rrnA and rrnB) have also been deposited in GenBank (accession numbers AB159557 and AB159558; similarity values of 98·6 % for the type A sequence and the rrnA sequence and 99·8 % for the type B sequence and the rrnB sequence). This organism is also a mesophilic, spore-forming, syntrophic propionate-oxidizing bacterium (H. Imachi, personal communication). The closest relative which has been studied in more detail is Cryptanaerobacter phenolicus LR7.2T (95·6 and 96·7 % sequence similarity to the type A and type B sequences, respectively), an anaerobic bacterium that transforms phenol and 4-hydroxybenzoate into benzoate (Juteau et al., 2005). This organism was not tested for syntrophic growth on propionate or other compounds. Another related bacterium, the thermophilic propionate-oxidizer Pelotomaculum thermopropionicum SIT, does grow syntrophically on propionate and is also unable to reduce sulfate (Imachi et al., 2002). Within the genus Desulfotomaculum, another thermophilic, spore-forming, syntrophic propionate-oxidizing bacterium (strain TPOT) was also described recently (Plugge et al., 2002). Unlike these two thermophilic strains, we were not able to grow strain HHT axenically, despite testing all substrates that supported growth of other syntrophs. Wu et al. (1992) described a mesophilic, spore-forming, propionate-oxidizing bacterium. This strain (strain PT) was isolated in co-culture with Methanobacterium formicicum MFNT and was one of the prevalent syntrophic propionate oxidizers in the UASB granules they studied. Although somewhat shorter in length, cells of strain PT had the same characteristics as the bacterium described here; the strain produced endospores and was not able to reduce sulfate in the presence of propionate (Wu et al., 1992). Besides butyrate, strain PT was not tested for growth on other substrates and, unfortunately, no phylogenetic information is available for this organism. Using stable isotope probing, it was demonstrated that, in addition to Syntrophobacter spp. and Smithella spp., Pelotomaculum spp. are important in propionate oxidation in anoxic paddy soil (Lueders et al., 2004).
Based on its physiological properties and 16S rRNA gene sequence similarity to Pelotomaculum thermopropionicum SIT, we propose that strain HHT represents a novel species in the genus Pelotomaculum, Pelotomaculum schinkii sp. nov. A consensus tree based on distance-matrix and parsimony analysis is depicted in Fig. 2.
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Unlike Pelotomaculum thermopropionicum, Pelotomaculum schinkii is mesophilic and converts only propionate in syntrophic association with hydrogenotrophic methanogens. All other characteristics of the genus as described by Imachi et al. (2002) remain unaffected.
Description of Pelotomaculum schinkii sp. nov.
Pelotomaculum schinkii (schin'ki.i. N.L. gen. n. schinkii named after Bernhard Schink, who studied several syntrophic conversions. The first spore-forming syntrophic bacterium, Syntrophospora bryantii, was isolated by his group).
Gram-positive rods, 1x2–2·5 µm in size, which produce spherical endospores in the late exponential phase; non-motile, strictly anaerobic. Grows syntrophically on propionate with methanogens that utilize both hydrogen and formate, such as Methanospirillum hungatei JF-1T and Methanobacterium formicicum MFNT. No organic supplements are required for growth, but yeast extract (0·1 %) and fumarate (10–20 mM) enhance growth. Habitat: anaerobic mesophilic granular sludge.
The type strain, HHT (=ATCC BAA-615T=DSM 15200T), was isolated from freeze-dried granular sludge from a UASB reactor treating sugar beet waste in co-culture with Methanospirillum hungatei JF-1T.
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ACKNOWLEDGEMENTS |
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