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1 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China
2 Graduate School of the Chinese Academy of Sciences, Beijing 100080, PR China
Correspondence
Xiuzhu Dong
dongxz{at}sun.im.ac.cn
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Published online ahead of print on 3 June 2005 as DOI 10.1099/ijs.0.63807-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Proteiniphilum acetatigenes TB107T is AY742226.
A comparison of the cellular fatty acid compositions of Proteiniphilum acetatigenes TB107T and its phylogenetic relatives is available as a supplementary table in IJSEM Online.
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). Neither syntrophic propionate-degrading bacteria nor methanogens utilize saccharides and proteinaceous substances, but can utilize short-chain organic acids or inorganics such as CO2. Pyruvate is an important intermediate not only in the syntrophic degradation of propionate, but also in the dissimilation of glucose and some amino acids. During an investigation of syntrophic propionate-degrading bacteria from methanogenic environments, some propionate-degrading tricultures were isolated. These tricultures degraded propionate to acetate and methane, and included three different micro-organisms; firstly, an egg-shaped, syntrophic, propionate-degrading bacterium (Chen et al., 2005); secondly, Methanobacterium formicicum-like cells; and finally, an unidentified rod-shaped bacterium. The rod-shaped bacterium on its own did not consume propionate or synthesize methane; however, it accelerated the propionate-degradation rate of the triculture. The purified rod-shaped bacterial strains utilized pyruvate and produced acetate and propionate from proteinaceous materials, but not glucose or other sugars. Phylogenetically the strains were affiliated to the Cytophaga–Flavobacterium–Bacteroides (CFB) group; however, they were only distantly related to recognized genera in this cluster. Hence, a new genus was designated based on the distinctive phenotypic and phylogenetic characteristics of the two strains.
Strains TB107T and TB6-6 were isolated in pre-reduced peptone-yeast extract (PY) medium (Holdeman et al., 1977) from methanogenic propionate-degrading mixtures by serial dilution and the Hungate roll-tube technique (Hungate, 1969). Single colonies were picked, and transferred to the same broth and incubated at 37 °C for 2 days. The roll-tube procedure was repeated several times until a pure culture was obtained. Culture purity was also checked by microscopic examination. Routine cultivation was in PY broth in anaerobic tubes (18x180 mm) sealed with butyl rubber stoppers under a gaseous atmosphere of 100 % N2 (100 kPa) at 37 °C.
Substrate utilization studies were performed in a basal medium containing various substrates: peptone, yeast extract and tryptone at 0·2 % (final concentration); sugars, fatty acids and alcohols at 20 mM (final concentration); and amino acids at 10 mM (final concentration). The basal medium contained (l–1): 1 g NH4Cl, 0·3 g K2HPO4, 0·3 g KH2PO4, 0·6 g NaCl, 0·1 g CaCl2.2H2O, 0·2 g MgCl2, 0·1 g KCl and 1 mg resazurin. The pH was adjusted to 7 with 1 M NaOH and cultivation conditions were as described above.
Cell morphology was examined under a light microscope (Olympus BH-2) and an electron microscope (Hitachi H-600A). For electron microscopy studies, bacterial cells grown in PY at 37 °C for 2 days were negatively stained with uranyl acetate. For ultrathin-section examination of the cell wall, bacterial cells were fixed with osmic acid and embedded in araldite; the samples were then sliced and stained with lead citrate (Reynolds, 1963).
The generation time of the strains was determined by monitoring the OD600 of the PY culture at 37 °C at 1 h intervals up to 72 h. Temperature profiles were determined in PY broth by using a water bath (Guangming medical instrument plant, Beijing) at temperatures of 15 to 55 °C, at 1 °C intervals. The pH range for growth was determined in PY broth at various pH values adjusted with HCl or NaOH (1 mol l–1). Growth was determined by measuring the OD600 of cultures at 1, 3 and 7 days. Biochemical traits were determined using both conventional methods (Holdeman et al., 1977) and the API 50 CH system (bioMérieux). All of the tests were performed in duplicate. Short-chain fatty acids and gases of fermentation were detected by using a gas chromatograph (GC-14B; Shimadzu), as described previously (Chen & Dong, 2004).
Genomic DNA was extracted and purified by using the method of Marmur (1961). The G+C content of the DNA was determined by the thermal denaturation method (Marmur & Doty, 1962) using a DU800 spectrophotometer (Beckman) with Escherichia coli K-12 as the reference. The 16S rRNA gene was amplified and sequenced according to Chen & Dong (2004). Sequencing was performed by Sangon Biological Engineering Technology Service, Shanghai, China, using ABI PRISM Big Dye Terminator cycle sequencing ready reaction kits (Perkin Elmer) and an ABI PRISM 377XL DNA sequencer. The 16S rRNA gene sequence of strain TB107T was submitted to GenBank and EMBL to search for similar sequences using the BLAST algorithm. The best matching sequences were retrieved from the database and aligned, and similarity analysis was performed using the program CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were constructed by using neighbour-joining, maximum-likelihood and maximum-parsimony methods implemented in the program MEGA2 (Kumar et al., 2001) and the PHYLIP package (Felsenstein, 1993). The resultant tree topologies were evaluated by bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings.
Cellular fatty acids were extracted, methylated and analysed using the standard MIDI (Microbial Identification) system (Miller, 1982; Sasser, 1990).
Cells of strains TB107T and TB6-6 were rod-shaped (0·6–0·9x1·9–2·2 µm), occurring singly or in pairs, and were motile by means of two peritrichous flagella (Fig. 1a). The cell wall was Gram-negative, as confirmed by the KOH lysis test (Smibert & Krieg, 1994) and ultrathin-section electron microscopy of strain TB107T (Fig. 1b). No spores were observed and resistance to treatment at 80 °C for 10 min was not observed. Colonies on PY agar were white, smooth, circular, entire, translucent and slightly convex, reaching 1·5 mm in diameter after cultivation at 37 °C for 72 h.
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The two strains exhibited almost identical physiological and biochemical profiles determined using conventional methods as well as the API 50 CH system. Both isolates hydrolysed aesculin and starch, but not gelatin. NH3 was produced from yeast extract, peptone and L-arginine, but indole was not produced. H2S was not produced from peptone or thiosulfate.
The similarity between the partial 16S rRNA gene sequences (500 bp) of strains TB107T and TB6-6 was 99·6 % and their G+C contents were 46·6 and 48·9 mol%, respectively. All of the above results indicate the single species status of the two isolates.
To ascertain the phylogenetic position of the new isolates, the complete 16S rRNA gene sequence (1523 bp) of strain TB107T was compared with the most similar sequences retrieved from GenBank. On the basis of a consensus 1424 bp of the 16S rRNA gene sequence, a phylogenetic tree rooted with Capnocytophaga gingivalis ATCC 33624T and Capnocytophaga haemolytica JCM 8565T was constructed (Fig. 2). Phylogenetic analysis showed that strain TB107T belonged to the CFB group (Paster et al., 1994) and clustered with Dysgonomonas species (89·6–90·6 % sequence similarity). Bootstrap resampling showed that this relationship was statistically significant (97 % recovery in 1000 resamplings). Other remotely related taxa included the genera Bacteroides (85–87 % sequence similarity), Porphyromonas (84–88 % sequence similarity), Prevotella (79–85 % sequence similarity) and Tannerella forsythensis (89·3 % sequence similarity). Based on the sequence divergence, it was evident that the new isolates could not be assigned to any of the described genera in this cluster.
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). They differed from Dysgonomonas species by the latter's cell shape of coccobacilli, facultative anaerobic growth, lower genomic DNA G+C content (38 mol%) and the ability to ferment glucose. Unlike the non-motile fusiform species T. forsythensis, the new isolates did not produce phenylacetic acid and butyric acid from glucose. The novel species differed from Bacteroides distasonis and Bacteroides merdae by the latter being non-motile and able to produce a large amount of succinic acid during glucose fermentation.
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).
An obligate syntrophic propionate-degrading bacterium, Syntrophobacter sulfatireducens DSM 16706T, was also isolated from the same upflow anaerobic sludge blanket (UASB) reactor as strain TB107T (Chen et al., 2005). This syntrophic strain converted 20 mM propionate to acetate and methane in co-culture with M. formicicum DSM 1535T in 50–60 days, with a propionate-degradation rate of 0·75 mM per day during the exponential phase. When in the triculture with strain TB107T, 20 mM propionate could be degraded in less than 30 days and the degradation rate was accelerated to about 1·25 mM per day. It appeared that acceleration of the propionate-degradation rate of the syntrophic propionate co-culture by strain TB107T was due to the provision of unknown nutrient factors or the depletion of pyruvate.
Description of Proteiniphilum gen. nov.
Proteiniphilum (Pro.tei'ni.phi.lum. N.L. neut. n. proteinum protein; Gr. adj. philos loving; N.L. neut. n. Proteiniphilum protein loving).
Gram-negative, motile, non-spore-forming rods. Obligately anaerobic. Microaerophilic or aerobic growth does not occur. Cellular fatty acids mainly consist of iso-branched fatty acids, predominantly anteiso-C15 : 0. Mesophilic. Grow at 20–45 °C. Chemoorganotrophic. Cytochrome oxidase and catalase are not produced. Proteolytic. Yeast extract and peptone can be used as energy sources. Carbohydrates and alcohols are not used. Gelatin is not hydrolysed. Not resistant to 20 % bile. The major fermentation products from PY are acetic acid and propionic acid. Nitrate is not reduced. The G+C contents of the genomic DNA of the known strains of the type species are 46·6 and 48·9 mol%. Only one species, Proteiniphilum acetatigenes, is described so far; this species has been designated the type species.
Description of Proteiniphilum acetatigenes sp. nov.
Proteiniphilum acetatigenes (a.ce'ta.ti.gen.es. N.L. acetas -atis acetate; Gr. v. gennao produce; N.L. part. adj. acetatigenes acetate-producing).
Morphology and general characteristics are as described for the genus. Cells are 0·6–0·9x1·9–2·2 µm. Colonies on PY agar are circular, slightly convex, white, translucent, and reach 1·5 mm in diameter after 3 days incubation at 37 °C. Optimum growth occurs at 37 °C. The pH range for growth is 6·0–9·7, with optimum growth occurring at pH 7·5–8·0. In addition to yeast extract and peptone, pyruvate, glycine and L-arginine can be used as carbon and energy sources. Weak growth is observed with tryptone, L-serine, L-threonine and L-alanine. Acetic acid is the main product from fermentation of yeast extract, peptone, pyruvate and L-arginine. Propionic acid is also produced from yeast extract. The following substrates are not used: L-histidine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-valine, L-glutamine, tryptophan, L-tyrosine, L-isoleucine, L-proline, L-aspartate, L-cysteine, L-arabinose, cellobiose, aesculin, D-fructose, D-galactose, D-glucose, glycogen, inulin, D-lactose, D-maltose, mannose, melibiose, raffinose, rhamnose, ribose, sucrose, salicin, sorbose, starch, trehalose, D-xylose, adonitol, amygdalin, dulcitol, erythritol, inositol, mannitol, sorbitol, ribitol, methanol, ethanol, 1-propanol, citrate, fumarate, malate, succinate, malonate, hippurate, sodium gluconate, butane diacid, 
-hydroxybutyric acid, phenylacetic acid, cellulose and xylan. Milk is not curdled. Starch and aesculin are hydrolysed. Indole is not produced. Urease, lecithinase and lipase are not produced. Methyl red and Voges–Proskauer tests are negative. H2S is not produced from peptone or thiosulfate. NH3 is produced from yeast extract, peptone and L-arginine. The major cellular fatty acids are anteiso-C15 : 0 (46·21 %), C15 : 0 (8·90 %), iso-C17 : 0 3-OH (5·93 %) and anteiso-C17 : 0 (5·15 %).
The type strain is TB107T (=JCM 12891T=AS 1.5024T), which was isolated from the granule sludge of a UASB reactor treating brewery wastewater.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chen, S., Liu, X. & Dong, X. (2005). Syntrophobacter sulfatireducens sp. nov., a novel syntrophic, propionate-oxidizing bacterium isolated from UASB reactors. Int J Syst Evol Microbiol 55, 1319–1324.
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