Desulfovirga Adipica Gen. Nov., Sp. Nov., an Adipate-Degrading, Gram-Negative, Sulfate-Reducing Bacterium

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 639–644 Printed in Great Britain

Desulfovirga adipica gen. nov., sp. nov., an adipate-degrading, Gram-negative, sulfate-reducing bacterium

Kazuhiro Tanaka,1 Erko Stackebrandt,2 Shigehiro Tohyama3 and Tadashi Eguchi3

Author for correspondence: Kazuhiro Tanaka. Tel\Fax: j81 298 61 6083. e-mail: ktanaka!nibh.go.jp

1 Applied Microbiology A novel, mesophilic, Gram-negative bacterium was isolated from an anaerobic Department, National digestor for municipal wastewater. The bacterium degraded adipate in the Institute of Bioscience and Human-Technology, presence of sulfate, sulﬁte, thiosulfate and elemental sulfur. (E)-2- Higashi 1-1, Tsukuba, Hexenedioate accumulated transiently in the degradation of adipate. (E)-2- Ibaraki 305-8566, Japan Hexenedioate, (E)-3-hexenedioate, pyruvate, lactate, C1–C12 straight-chain fatty 2 Deutsche Sammlung von acids and C2–C10 straight-chain primary alcohols were also utilized as electron Mikroorganismen und donors. 3-Phenylpropionate was oxidized to benzoate. The GMC content of the Zellkulturen GmbH, Mascheroder Weg 1b, DNA was 60 mol%. 16S rDNA sequence analysis revealed that the new isolate D-38124 Braunschweig, clustered with species of the genus Syntrophobacter and Desulforhabdus Germany amnigenus. Strain TsuAS1T resembles Desulforhabdus amnigenus DSM 10338T 3 Department of Chemistry with respect to the ability to utilize acetate as an electron donor and the and Materials Science, inability to utilize propionate without sulfate in co-culture with Tokyo Institute of T T Technology, O-okayama, Methanospirillum hungatei DSM 864. Strains TsuAS1 and DSM 10338 form a Meguro-ku, Tokyo ‘non-syntrophic subcluster’ within the genus Syntrophobacter. Desulfovirga 152-8551, Japan adipica gen. nov., sp. nov. is proposed for the newly isolated bacterium, with strain TsuAS1T (l DSM 12016T) as the type strain.

Keywords: adipic acid, sulfate reduction, anaerobic digestor, mesophilic bacterium, Desulfovirga adipica

INTRODUCTION METHODS Source of the isolate and micro-organisms. Strain TsuAS1T Adipic acid is a natural product contained in the lichen was isolated from an anaerobic digestor of a municipal Cetraria islandica (Solberg, 1986) and in leaves of the wastewater treatment centre in Tsuchiura, Ibaraki, Japan, plant Limonia acidissima (Das & Thakur, 1989). This and was deposited in the Deutsche Sammlung von Mikro- acid is also synthesized industrially as a raw material to organismen und Zellkulturen, Braunschweig, Germany, as synthesize polymers such as ‘nylon 6,6’ and poly- strain DSM 12016T. Methanospirillum hungatei strain JF1 urethanes, and plasticizers such as di(2-ethylhexyl)- (DSM 864) was provided by Y. Kamagata (NIBH, Japan). adipate, all of which are important industrial products. Chemicals. (E)- and (Z)-2-hexenedioates were synthesized as This acid is known to be degraded aerobically, for follows. γ-Butyrolactone was treated with p-toluenesulfonic example by Pseudomonas aeruginosa, Acidovorax dela- acid in ethanol to give ethyl 4-hydroxybutyrate. Oxidation fieldii and ‘Pseudomonas saccharophila’ (Palleroni, of ethyl 4-hydroxybutyrate by DMSO-oxalyl chloride\ 1984). To the authors’ knowledge, however, its an- triethylamine afforded an intermediary aldehyde, which was aerobic degradation by a pure culture of a sulfate subjected to the Wittig reaction with (carbethoxymethylene)- reducer has not been documented. Here, we describe triphenylphosphorane in methanol to give a mixture of the isolation and characterization of a novel Gram- diethyl (E)- and (Z)-2-hexenedioates (about 2:1). After negative, mesophilic and sulfate-reducing strain chromatographic separation over silica gel, each isomer was capable of growing by completely oxidizing adipate. finally hydrolysed with lithium hydroxide, followed by acid treatment to give the desired (E)- and (Z)-2-hexenedioic acids...... The GenBank/DDBJ accession number for the 16S rDNA gene sequence of (E)-2-Hexenedioic acid: colourless powder; m.p. 179– " strain TsuAS1T is AJ237605. 180 mC; H NMR (500 MHz, DMSO-d')d2n37 (m, 4H), 5n76

01226 # 2000 IUMS 639 K. Tanaka and others

(broad d, J 15 6 Hz, 1H), 6 80 (m, 1H), 12 13 (broad s, Light microscopy. Cells were observed with an Olympus BH- "$ l n n n 2H); C NMR (125 MHz, DMSO-d')d26n9, 32n1, 122n5, 2 phase-contrast microscope. Micrographs were taken after 147n4, 167n1, 173n6; IR (KBr), 2923, 1689, 1647, 1421, 1321, mounting cultures on glass slides coated with 2% agar −" 1214 cm . Elemental analysis. Calculated for C'H)O%:C, (Pfennig & Wagener, 1986). 50 00; H, 5 59. Found: C, 50 29; H, 5 77. (s, singlet; d, n n n n Electron microscopy. Cells on the grid were negatively stained doublet; m, multiplet) with 1% uranyl acetate. Photographs were taken using a (Z)-2-Hexenedioic acid: colourless powder; m.p. 78–80 C; JEM-1010 transmission electron microscope (JEOL) at " m H NMR (500 MHz, DMSO-d')d2n33(t, J l 7n4 Hz, 2H), 100 kV. 2 74 (dq, J 1 7 and 7 4 Hz, 2H), 5 72 (dt, J 11 5 and n l n n n l n Analysis of adipate and metabolic products. Determination 1 7 Hz, 1H), 6 19 (dt, J 11 5 and 7 4 Hz, 1H), 12 14 (broad n "$ n l n n n of adipate, (E)-2-hexenedioate and (Z)-2-hexenedioate was s, 2H); C NMR (125 MHz, DMSO-d )d242, 33 1, 121 3, ' n n n carried out as follows. Culture fluid (1 0 ml) was centrifuged 147 8, 167 2, 173 9; IR (KBr), 2939, 1701, 1649, 1604, 1450, n n n n " in a 1 5 ml polypropylene tube. Then, 0 45 ml supernatant 1419, 1389, 1300, 1261, 1226, 1160 cm− . Elemental analysis. n n was transferred to a new 1 5 ml polypropylene tube and Calculated for C H O :C,5000; H, 5 59. Found: C, 50 24; n ' ) % n n n acidified with 50 µl1MHPO , followed by analysis of the H, 5 77. (s, singlet; d, doublet; t, triplet; dt, double triplet;dq, $ % n acidified supernatant by HPLC using a 6 150 mm Shim- double quartet) i pack CLC-ODS column (Shimadzu) at 40 mC, 9 mM −" Hexanoic acid and heptanoic acid were purchased from H$PO%\2n5% (v\v) methanol as eluant at 1 ml min , and a Wako and Aldrich, respectively. UV detector at 210 nm. Benzoate was determined similarly, except 3 mM H PO \49% methanol was as eluant (Tanaka Culture conditions. Strain TsuAS1T was cultured using the $ % et al., 1991). Sulfide was determined spectrophotometrically Hungate technique (Bryant, 1972). A basal medium con- via a method yielding methylene blue (Cline, 1969). tained 2 mM Na#S, 1 µM resazurin, 30 mM NaHCO$, 1mMNH%Cl, 1 mM KH#PO%, 1 mM MgCl#, 1 mM CaCl#, Pigment analysis. Desulfoviridin was determined as de- 10 µM FeCl#,1µM CoCl#,1µM MnCl#,1µM ZnCl#, scribed by Postgate (1956). Cytochromes were identified by 100 nM H$BO$, 100 nM NiCl#, 100 nM Na#MoO%, 100 nM taking redox difference spectra of dithionite-reduced minus AlCl$,10nMNa#SeO$,10nMNa#WO%, 10 nM CuCl#, air-oxidized cell-free extracts with a Shimadzu UV-1600PC 100 nM biotin, 100 nM 4-aminobenzoic acid, 100 nM pan- spectrophotometer. tothenic acid, 100 nM pyridoxine, 100 nM nicotinic acid, Isolation of DNA and base composition. Cells were lysed by 100 nM thiamine, 100 nM lipoic acid, 100 nM folic acid, treatment with achromopeptidase and SDS (Ezaki et al., 100 nM riboflavin and 100 nM vitamin B , under an "# 1990). DNA was isolated from the resultant lysate (Marmur, atmosphere of N \CO (80:20, v\v). The medium was # # 1961) and base composition of DNA was determined using prepared using a Widdel flask (Widdel & Bak, 1992). The pH HPLC (Tamaoka & Komagata, 1984; Kamagata et al., of the basal medium was 6 9–7 1 unless otherwise stated. n n 1992). When pH dependence was studied, the N#\CO# ratio was varied in a range between 100:0 and 50:50. For sulfate 16S rDNA sequence determination. Genomic DNA extrac- reduction, 5 mM sodium sulfate was added unless otherwise tion, PCR-mediated amplification of the 16S rDNA and stated. A time course study was carried out using half-filled sequencing of the PCR products were performed as de- 130 ml serum bottles in duplicate. Substrate tests were scribed by Rainey et al. (1996). The sequence reaction carried out using half-filled 20 ml Pyrex tubes in duplicate mixtures were electrophoresed using a model 373A auto- for water-soluble substrates. Lipophilic substrates such as matic DNA sequencer (Applied Biosystems). decanoic acid (5 mM), undecanoic acid (5 mM), dodecanoic Phylogenetic analysis. The 16S rDNA sequence of strain acid (5 mM), tridecanoic acid (5 mM), 1-hexanol (10 mM), T 1-heptanol (5 mM), 1-octanol (5 mM), 1-nonanol (5 mM), TsuAS1 was aligned to the sequences of a general selection 1-decanol (5 mM), 1-undecanol (5 mM), 2-phenylethanol of different bacteria in the DSMZ identification database, δ (50 mM), 2-phenylpropanol (50 mM) and indole (10 mM) and analysed against members and relatives of the -subclass Proteobacteria et al were prepared in sterile stock solutions in decahydro- of the using the ae2 editor (Maidak ., naphthalene at the concentrations indicated. A test tube for 1997). Evolutionary distances were calculated by the method of Jukes & Cantor (1969). Phylogenetic dendrograms were the lipophilic substrate test was prepared to contain 2n0ml substrate solution and 19 2 ml basal medium in a 25 mm constructed according to the method of DeSoete (1983) and n the neighbour-joining method contained in the  (o.d.)i150 mm order-made screw-capped tube plugged with a Teflon-coated butyl rubber stopper, which was then package (Felsenstein, 1993). inoculated with 0n8 ml preculture to give a final aqueous volume of 20n0 ml. In this case, one-tenth of the substrate RESULTS concentration in decahydronaphthalene is given in par- entheses in Results to make it easy to compare with results Enrichment and isolation with hydrophilic substrates. The inoculum size was 4% (v\v), as with hydrophilic substrates. A pure culture was Liquid medium containing 1 mM adipate and 5 mM obtained by application of the agar dilution method sulfate in tubes was inoculated with sludge, mud and (Pfennig, 1978). To test the capacity of strain TsuAS1T for sand samples from anaerobic digestors, a creek, a syntrophic growth on each of adipate, ethanol, 1-propanol, pond and a lake, and was incubated at 30 mC. Slight lactate, propionate and butyrate without sulfate in co- growth was observed after 3 months with a tube culture with Methanospirillum, strain TsuAS1T was pre- inoculated with a sludge sample from an anaerobic cultured on each of the substrates in the presence of sulfate. digestor of the municipal wastewater treatment centre Growth determination. Cell density was determined with a in Tsuchiura, Ibaraki, Japan. The obtained enrichment Shimadzu UV-1600PC spectrophotometer at 600 nm using culture could be subcultured and was subjected to 1 cm cuvettes. serial liquid dilution with liquid medium for partial

640 International Journal of Systematic and Evolutionary Microbiology 50 Desulfovirga adipica gen. nov., sp. nov.

hibited growth. In the presence of 2 mM sulﬁde, growth was inhibited by the addition of 2 mM cysteine or thioglycolic acid.

Metabolism With adipate provided as electron donor, strain TsuAS1T utilized sulfate, sulfite, thiosulfate and elemental sulfur (weak growth) as electron acceptors. Nitrate was not utilized as electron acceptor. Con- sumption of 1 mM adipate and 5 mM sulfate (excess), lead to the production of 2n90p0n06 mM H#S. Electron recovery was 89p2% as calculated based on complete ...... oxidation of adipate. Trace amounts (about 6 µM) of Fig. 1. Phase-contrast micrograph of cells of strain TsuAS1T in (E)-2-hexenedioate accumulated transiently during the mid-exponential phase. Bar, 10 µm. oxidation of adipate, while (Z)-2-hexenedioate was not detected (see Fig. 3). T −" Electron donors utilized by strain TsuAS1 include purification. Yeast extract at 0n5gl enhanced growth adipate (1 mM), (E)-2-hexenedioate (1 mM), (E)-3- of the partially purified enrichment culture. Following hexenedioate (1 mM), pyruvate (2 mM), lactate agar dilution, lens-shaped white colonies appeared (2 mM), formate (10 mM), acetate (2 mM), propi- after 4 weeks. A colony in the highest dilution tube was onate (2 mM), butyrate (1 mM), valerate (1 mM), transferred into liquid medium. Purity of the culture T caproate (1 mM), heptanoate (1 mM), octanoate (designated strain TsuAS1 ) was confirmed by cell (0n5 mM), nonanoate (0n5 mM), decanoate (0n5 mM), morphology under the phase-contrast microscope and undecanoate (0n5 mM), dodecanoate (0n5 mM), iso- by the lack of growth on any of glucose (2 mM), −" −" butyrate (1 mM), isovalerate (1 mM), isocaproate peptone (1 g l ) and yeast extract (1 g l ). (1 mM), 3-phenylpropionate (0n5–2 mM), ethanol (2 mM), 1-propanol (2 mM), 1-butanol (1 mM), 1- Morphology pentanol (1 mM), 1-hexanol (1 mM), 1-heptanol T (0n5 mM), 1-octanol (0n5 mM), 1-nonanol (0n5 mM) Cells of strain TsuAS1 were Gram-negative, straight and 1-decanol (0n5 mM). The amount of H#S produced to slightly curved short rods, single or in pairs, with was close to the amount expected for complete rounded ends having the shape of a rice grain (Fig. 1). oxidation of substrates, except with decanoate, un- The majority of the cells were 2n2–4n0 µm long and decanoate, dodecanoate and 1-decanol (stoichiometry 0n8–2n0 µm wide (Fig. 1). Cells were motile and had a not studied with these substrates). 3-Phenylpropionate single polar flagellum (Fig. 2). Spores were never T was almost stoichiometrically oxidized to benzoate observed in the culture of strain TsuAS1 under the (98p3 mol%). microscope and pasteurized digested sludge from T Electron donors not utilized for growth by strain which strain TsuAS1 was isolated did not yield micro- T organisms which could utilize adipate in the presence TsuAS1 include H#\CO# (80:20, v\v), (Z)-2-hexene- of sulfate over a 7 month period. dioate (1 mM), methylmalonate (2 mM), malonate (2 mM), succinate (2 mM), glutarate (1 mM), pimelate (1 mM), suberate (1 mM), muconate (1 mM), fumar- Physiological characterization ate (2 mM), maleate (2 mM), -malate (2 mM), - Growth of the new isolate was observed between malate (2 mM), tridecanoate (0n5 mM), benzoate (1 mM), cinnamate (0 5 mM), phenylacetate (1 mM), pH 6n6 (not tested below pH 6n6) and pH 7n4 with an n 2-phenylpropionate (0 5 mM), 4-phenylbutyrate optimum around pH 7n0. No growth occurred at n T (0 5 mM), 6-phenylhexanoate (0 5 mM), cyclohexane- pH 7n6. Strain TsuAS1 grew at 20–36 mC with an n n carboxylate (1 mM), methanol (2–5 mM), 2-methoxy- optimum around 35 mC. At temperatures of 15 and ethanol (2 mM), ethylene glycol (2 mM), 1,3-propane- 37 mC, no growth occurred. Under optimal conditions, T −" diol (2 mM), 1,4-butanediol (2 mM), 1,5-pentanediol strain TsuAS1 grew at a rate of 0n06–0n07 d (doub- T (2 mM), 1-undecanol (0 5 mM), 2-phenylethanol ling time l 10–12 d). Growth of strain TsuAS1 was n enhanced by yeast extract and the growth yield was (5 mM), 2-phenylpropanol (5 mM), phenol (1 mM), −" catechol (1 mM), glucose (1 mM), fructose (1 mM), optimal at 0n5–1 g yeast extract l as estimated from xylose (1 mM), mannitol (1 mM), acetone (2 mM) and OD'!! measurements (data not shown). In the presence of 100 µM EDTA, the yield was optimal at 0 02 g indole (1 mM). " n yeast extract l− (a comparison between the optimal growth yield with and without EDTA was not carried DNA base composition out). Nitrilotriacetate and citrate were not as effective as EDTA. Addition of 70 mM sodium chloride in- The GjC content of the DNA was 59n9p0n5mol%.

International Journal of Systematic and Evolutionary Microbiology 50 641 K. Tanaka and others

...... Fig. 2. Electron micrograph of a single, ﬂagellated cell of strain TsuAS1T negatively stained with 1% uranyl acetate. Bar, 1 µm.

8 0·08 between 93n6 and 94n5%. The bootstrap value determined for the branching point of the new isolate is 50%, hence without statistical signiﬁcance.

6 0·06 DISCUSSION M); pH l

S (mM); T

2 Among strains TsuAS1 , Syntrophobacter sp. strain HP1.1 (Zellner et al., 1996), Syntrophobacter fumar- 4 0·04 600 oxidans DSM 10017T (Harmsen et al., 1998), Syn- OD trophobacter pfennigii DSM 10092T (Wallrabenstein et al., 1995), Desulforhabdus amnigenus DSM 10338T (Oude Elferink et al., 1995) and Syntrophobacter

)-2-hexenedioate ( 2

Adipate (mM); H 0·02 T

E wolinii DSM 2805 (Boone & Bryant, 1980), only ( strains TsuAS1T and Desulforhabdus amnigenus DSM 10338T do not grow in co-culture with Methano- spirillum hungatei on propionate and oxidized acetate. 510152025 In spite of the physiological similarity and a moderate Time (d) phylogenetic relationship these two strains do not form a phylogenetically consistent ‘non-syntrophic ...... subcluster’ within the genus Syntrophobacter. How- Fig. 3. Time course of anaerobic oxidation of adipate by strain ever, the position of Syntrophobacter wolinii DSM T TsuAS1 . Two bottles showed similar proﬁles; data from one 2805T as the deepest branching strain of the genus bottle are shown. X,pH;$ (dotted line), OD600 ; >,(E)-2- hexenedioate; $ (solid line), H S; , adipate. Syntrophobacter is not supported by a high bootstrap 2 value, indicating a need for future extension of the database so that the non-syntrophic members may fall outside the radiation of the genus Syntrophobacter.It Pigments seems reasonable to consider that strain TsuAS1T Desulfoviridin was not detected in cell-free extract of represents a new species in a new genus, Desulfovirga strain TsuAS1T. A redox diﬀerence spectrum of adipica gen. nov., sp. nov., in the δ-subclass of the dithionite-reduced minus air-oxidized cell-free extract Proteobacteria. had peaks at 421, 523 and 552 nm, indicating the At the moment it is unknown whether the transient presence of cytochrome c. accumulation of (E)-2-hexenedioate during batch culture growth of strain TsuAS1T on adipate and sulfate, Phylogenetic analysis and the ability of the strain to utilize (E)-2-hexenedioate as electron donor, indicates that adipate is The phylogenetic analysis of the 1524 nt 16S rDNA degraded via (E)-2-hexenedioate, i.e. by β-oxidation. sequence revealed that strain TsuAS1T clusters with Syntrophobacter sp. strain HP1.1, Syntrophobacter Concerning growth of strain TsuAS1T, colonies ap- fumaroxidans DSM 10017T, Syntrophobacter pfennigii peared after 4 weeks in spite of the doubling time of DSM 10092T, Desulforhabdus amnigenus DSM 10338T 10–12 d. It seems that the strain grows faster in agar and Syntrophobacter wolinii DSM 2805T within the δ- medium than in the liquid medium. In addition, it is subclass of the Proteobacteria (see Fig. 4). Strain unknown why the yeast extract concentration for TsuAS1T appears almost equidistantly related to these optimal growth was lowered in the presence of 100 µM strains, sharing with them binary similarity values EDTA.

642 International Journal of Systematic and Evolutionary Microbiology 50 Desulfovirga adipica gen. nov., sp. nov.

Syntrophobacter sp. HP1.1

Synthrophobacter fumaroxidans DSM 10017T Synthrophobacter pfennigii DSM 10092T

Desulforhabdus amnigenus DSM 10338T Strain TsuAS1T (DSM 12016T)

Syntrophobacter wolinii DSM 2805T

Desulfacinum infernum DSM 9756T

Desulfomonile teidjei DSM 6799T

Desulfobacterium anilini DSM 4660T ‘Desulfoarculus baarsii’ M37317

Syntrophus buswellii DSM 2612T

Desulfobacterium catecholicum DSM 3882T Desulfobacterium macestii DSM 4194T

Desulfovibrio gigas ATCC 19364T Desulfobacterium cetonicum DSM 7267T Desulfobacterium indolicum DSM 3383T

Desulfobacterium phenolicum DSM 3384T

Desulfobacter vibrioformis DSM 8776T

Desulfobacterium autotrophicum DSM 3382T ...... Fig. 4. Phylogenetic dendrogram based on ‘Desulfobacterium niacini’ PM4 16S rDNA sequence showing the phylogenetic position of strain TsuAS1T and ‘Desulfobacterium vacuolatum’ M34408 related strains. Scale bar, 5 inferred 0·05 nucleotide substitutions per 100 nt.

Description of Desulfovirga gen. nov. uvate and lactate, C"–C* straight-chain fatty acids, C –C iso-fatty acids and C –C straight-chain primary Desulfovirga (de.sul.fo.vir ga. M.L. pref. desulfo de- % ' # * h alcohols are completely oxidized and 3-phenyl- sulfuricating; L. fem. n. virga twig, rod; Desulfovirga propionate is stoichiometrically oxidized to benzoate. sulfate-reducing rod). Straight-chain saturated C$–C) 1,ω-dicarboxylates are Rod-shaped cells. No spore formation. Gram-nega- not utilized except adipate. GjC content of DNA is tive. Strictly anaerobic. Chemo-organotrophic. 60 mol%. Habitat: anaerobic digestor of the mu- Sulfate, sulﬁte, thiosulfate and sulfur are utilized as nicipal wastewater treatment centre, Tsuchiura, T electron acceptors. Nitrate is not utilized. Desulfo- Ibaraki, Japan. Type strain is TsuAS1 (l DSM viridin has not been detected. Cytochrome c is present. 12016T). Propionate is not utilized in the absence of sulfate in co-culture with Methanospirillum hungatei. Growth ACKNOWLEDGEMENTS occurs at 20–36 mC. GjC content of DNA is 60 mol%. According to 16S rDNA analysis, Desulfo- We thank Professor K. Kakinuma (Tokyo Institute of virga represents a new genus, belonging to the δ- Technology, Japan) for encouragement to this work and subclass of the Proteobacteria. Type species is Desulfo- helpful discussions, and Professor H. G. Tru$ per (University of Bonn, Germany) for advice on the naming of strain virga adipica. T TsuAS1 . Description of Desulfovirga adipica sp. nov. REFERENCES Desulfovirga adipica (a.dihpi.ca. M.L. n. acidum Boone, D. R. & Bryant, M. P. (1980). Propionate-degrading adipinum adipic acid (adipate); M.L. fem. adj. adipica bacterium, Syntrophobacter wolinii sp. nov., gen. nov., from pertaining to adipic acid and the organism’s ability to methanogenic ecosystems. Appl Environ Microbiol 40, 626–632. degrade it). Bryant, M. P. (1972). Commentary on the Hungate technique for Cells are 0n8–2n0i2n2–4n0 µm with round ends, singly culture of anaerobic bacteria. Am J Clin Nutr 25, 1324–1328. or in pairs. Motile. In the presence of excess sulfate, Cline, E. (1969). Spectrophotometric determination of hydrogen- adipate, (E)-2-hexenedioate, (E)-3-hexenedioate, pyr- sulﬁde in natural waters. Limonol Oceanogr 14, 454–458.

International Journal of Systematic and Evolutionary Microbiology 50 643 K. Tanaka and others

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