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Faculty of Agriculture, Yamagata University, Wakaba-machi 1-23, Tsuruoka, Yamagata 997-8555, Japan
Correspondence
Atsuko Ueki
uatsuko{at}tds1.tr.yamagata-u.ac.jp
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6 and C18 : 17. Phylogenetic analysis based on 16S rRNA gene sequences placed both strains in the class Deltaproteobacteria. The closest recognized relative of strains Pro1T and Pro16 was Desulfobulbus mediterraneus with sequence similarities of 95.2 and 94.8 %, respectively. Based on phylogenetic, physiological and chemotaxonomic characteristics, strains Pro1T and Pro16 represent a novel species of the genus Desulfobulbus, for which the name Desulfobulbus japonicus is proposed. The type strain is Pro1T(=JCM 14043T=DSM 18378T) and strain Pro16 (=JCM 14044=DSM 18379) is a reference strain.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains Pro1T and Pro16 are AB110549 and AB110550, respectively.
Present address: Taisei Corporation, Naze-machi 344-1, Totsuka-ku, Yokohama, Kanagawa 245-0051, Japan. 
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). Major intermediates of anaerobic decomposition of organic matter such as acetate, propionate, butyrate and H2 serve as the most important electron donors for sulfate-reduction in environments and thus SRB contribute significantly to the mineralization of organic matter as well as to the Earth's sulfur cycle (Jørgensen, 1982; Sørensen et al., 1981). It is known that SRB often take a principal role in the oxidation of propionate in various anoxic environments (Parkes et al., 1993; Ueki et al., 1986). Of the propionate-oxidizing SRB from marine environments, species in the genera Desulfobacterium, Desulfonema and Desulfacinum oxidize propionate completely to carbon dioxide, whereas species in the genera Desulfobulbus, Desulforhopalus and Desulfofaba oxidize it incompletely to acetate (Kuever et al., 2005). In this study, two strains of propionate-oxidizing SRB, Pro1T and Pro16, that were isolated through enrichment culture with propionate as an electron donor from an estuarine sediment from the Japanese islands (Suzuki et al., 2007) were subjected to a comprehensive characterization.
Sediment cores were collected to a depth of 6 cm with a core sampler (5 cm in diameter) from sediment at a water depth of 2 m in the Niida river estuary in Sakata harbour, which is located on the coast of the Sea of Japan in the Japanese islands (38° 54.5' N 139° 50.6' E) on the 24 June 1999. Diluted (10–3–10–6) sediment samples (0.2 ml) were inoculated into liquid seawater medium containing 20 mM of sodium propionate as described below. Cultures showing sulfate-reduction by formation of a black precipitate in the medium were transferred to fresh medium containing the same electron donor. After two to four subcultures performed in the same manner, SRB were isolated from the enrichment culture using the anaerobic roll-tube method (Hungate, 1966) with sodium propionate as an electron donor. Several strains of SRB were obtained by picking up black colonies of SRB that appeared in agar roll-tubes after about one month of incubation. Strains Pro1T and Pro16 were finally obtained after several purification procedures through colony isolation by the anaerobic roll-tube method.
Two basal media (seawater medium and defined medium) were used in this study as described previously (Suzuki et al., 2007). The seawater medium, which was used for enrichment culture, contained (l–1 of seawater): 0.5 g KH2PO4, 0.3 g NH4Cl, 0.1 g yeast extract, 1 mg sodium resazurin, 10 ml trace element solution (modified from SL-10, described by Widdel et al., 1983), 1 ml B-vitamin solution (Widdel & Bak, 1992) and 0.5 g L-cysteine.HCl.H2O, as well as an appropriate electron donor. The pH was adjusted to 7.2–7.4 with 1 M NaOH. Agar (1.5 %, w/v; Difco) was added to the medium and used for the anaerobic roll-tube method for isolation and slant cultures with sodium propionate as an electron donor. The following medium, which was designated ‘defined medium’ in contrast to the seawater medium and used for the general physiological characterization of the strains, contained (l–1): 0.5 g KH2PO4, 1.0 g NH4Cl, 1.0 g Na2SO4, 2.0 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.5 g yeast extract, 1 mg sodium resazurin, 10 ml trace element solution, 1 ml B-vitamin solution, 30 g NaCl and 0.5 g L-cysteine.HCl.H2O (Nakamoto et al., 1996; Ueki et al., 1980; Widdel & Bak, 1992). The pH was adjusted to 7.2–7.4 with 1 M NaOH. Agar (Difco) (1.5 %, w/v) was added to the medium and used for the slant cultures with propionate as an electron donor. The concentration of sulfate in the defined medium was about 15 mM. Cultivation and transfer of the strains were performed under an O2-free N2 (100 %) atmosphere. The strains were cultivated at 30 °C, unless stated otherwise. The strains were maintained in slant cultures of seawater medium or defined medium with sodium propionate (20 mM) as an electron donor.
Flagella-staining was carried out according to Blenden & Goldberg (1965). Aerobic growth was examined in the presence of sodium propionate as an electron donor using the defined medium without L-cysteine.HCl.H2O and sodium resazurin. Catalase and oxidase activities of cells were tested as described by Akasaka et al. (2003a). The effects of NaCl concentration, temperature and pH on growth of the strains were examined in the presence of sodium propionate as an electron donor using the defined medium. Growth of the strains was monitored by measurement of OD660 with a spectrophotometer (U-1000; Hitachi).
Utilization of electron donors by the strains was determined using the defined medium containing each compound at a final concentration of 20 mM (fatty acids, amino acids and alcohols) or 10 mM (carbohydrates). H2 utilization was determined in the presence of sodium acetate (5 mM) with H2 in the atmosphere. Utilization of electron acceptors was determined with sodium propionate (20 mM) as an electron donor in a sulfate-free medium which contained the same concentrations of chloride in place of sulfate in the defined medium. Sodium sulfite (3 mM), sodium thiosulfate or disodium fumarate (20 mM each) were added to the sulfate-free medium as possible electron acceptors. Utilization of pyruvate, lactate, fumarate, malate (20 mM each), glucose and fructose (10 mM each) in the absence of electron acceptors in the medium was also determined using the sulfate-free medium. Fatty acids and amino acids were used in the form of sodium salts and added to the medium from sterilized stock solutions. Utilization of each electron donor or acceptor was determined by comparing the growth (OD660) in the presence or absence of each compound as well as by measurement of the concentration in the medium after cultivation.
Volatile fatty acids and alcohols were analysed by GC (G-5000 or 263-30; Hitachi), as described by Ueki et al. (1986). Non-volatile fatty acids and formate were analysed by HPLC (LC-10AD; Shimadzu) as described by Akasaka et al. (2003a). Reducing sugars were quantified by the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). Sulfate, sulfite and thiosulfate were analysed with an ion chromatograph (2000i; Dionex) as described by Nakamoto et al. (1996). Genomic DNA was extracted according to the method described by Kamagata & Mikami (1991). Extracted DNA was digested with P1 nuclease by using a Yamasa GC kit (Yamasa shoyu) and the G+C content was measured by HPLC (L-7400; Hitachi) equipped with a µBondpack C18 column (3.9x300 mm; Waters). Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987) and analysed by using a mass spectrometer (JMS-SX102A; JEOL). Whole-cell fatty acids (CFAs) were converted to methyl esters by saponification, methylation and extraction according to the method of Miller (1982). Methyl esters of CFAs were analysed by GC (HP6890, Hewlett Packard or G-3000, Hitachi) equipped with a HP Ultra2 column. CFAs were identified by equivalent chain-length (Miyagawa et al., 1979; Ueki & Suto, 1979) according to the protocol of TechnoSuruga Co., Ltd., based on the MIDI microbial identification system (Microbial ID) of Moore et al. (1994).
Extraction of DNA and PCR-amplification of the 16S rRNA gene of the novel strains were carried out according to the method described by Akasaka et al. (2003a, b) using a primer set, 27f and 1492r. The PCR-amplified 16S rRNA gene was sequenced by using a Thermo Sequenase Primer Cycle sequencing kit (Amersham Biosciences) and a DNA sequencer (4000L; Li-COR). Multiple alignments of the sequence with reference sequences in GenBank were performed with the BLAST program (Altschul et al., 1997). A phylogenetic tree was constructed with the neighbour-joining method (Saitou & Nei, 1987) by using the CLUSTAL W program (Thompson et al., 1994) as well as the maximum-likelihood program (DnaML) of the PHYLIP 3.66 package (Felsenstein, 2006). All gaps and unidentified base positions in the alignments were excluded before assemblages.
Strains Pro1T and Pro16 had almost the same physiological and chemotaxonomic characteristics. Cells of both strains were Gram-negative rods with rounded ends, 0.8–1.6 µm wide and 1.4–2.9 µm long. Cells usually occurred singly or in pairs (Fig. 1). Spore formation was not observed. Cells were motile by a single polar flagellum. Both strains made greyish and thin colonies on agar slants of the defined medium as well as the seawater medium.
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0.16 h–1 for both substrates) than with propionate (µ0.13 h–1) as an electron donor. Although growth rates with glucose (µ0.02 h–1) and fructose (µ0.07 h–1) were significantly lower than that found with propionate, final growth yields with these carbohydrates reached about twice (OD6602.0) those with propionate (OD660=0.8–1.0). Although almost all organic electron donors were oxidized to mainly acetate, propanol and butanol were oxidized to their corresponding carboxylic acids. Propionate (about 20 mM) produced by oxidation of propanol (20 mM) was not further oxidized in spite of the presence of unreduced sulfate (about 5 mM) in the medium. Glucose and fructose were oxidized to acetate with molar ratios of about 1 : 1 : 1.6–1.7 (carbohydrates : sulfate : acetate), which were slightly different from the theoretical ratio (1 : 1 : 2). The novel strains did not utilize acetate, butyrate, succinate, methanol, glycine, serine, aspartate, glutamate, cellobiose or sucrose as electron donors. Strain Pro1T did not use arabinose, ribose, xylose, galactose, mannose, rhamnose, sorbose, lactose or melibiose (utilization of these compounds was not determined for strain Pro16). Strains Pro1T and Pro16 utilized thiosulfate in addition to sulfate as an electron acceptor with propionate as an electron donor. Neither strain used sulfite or fumarate as electron acceptors. In the absence of electron acceptors, both strains oxidized pyruvate and lactate (about 5.5 mM each) and produced acetate (2.6–3.3 mM) and propionate (1.2–3.7 mM), while neither oxidized fumarate or malate. In the absence of electron acceptors, strain Pro1T oxidized small amounts of glucose and fructose (about 2 mM each) and produced trace amounts of acetate (0.2 mM and 0.5 mM, respectively), propionate (0.2 mM each) and H2.
The G+C contents of the genomic DNA of strains Pro1T and Pro16 were 48.6 and 46.0 mol%, respectively. The major respiratory quinone of both strains was menaquinone MK-5(H2). Strains Pro1T and Pro16 had almost the same content of CFAs with C15 : 0 (21.0 and 21.7 %, respectively), C16 : 0 (7.5 and 7.1 %), C17 : 16 (39.2 and 46.4 %) and C18 : 17 (12.4 and 11.4 %) as major CFAs for both strains.
Almost full-length 16S rRNA gene sequences were determined for both strains (Pro1T, 1485 bp; Pro16, 1479 bp). The sequence similarity between the two strains was 98.1 %. Based on phylogenetic analysis of the 16S rRNA gene sequence, strains Pro1T and Pro16 were affiliated with the class Deltaproteobacteria and formed a cluster in the family Desulfobulbaceae (Fig. 2). The closest relative of both strains found on the database was Desulfobulbus sp. BG25, isolated from salt marsh sediment, with gene sequence similarities of 98.5 and 98.4 % to strains Pro1T and Pro16, respectively. The closest recognized species to the novel strains were Desulfobulbus mediterraneus (Sass et al., 2002), with sequence similarities of 95.2 (sequence length compared, 1428 bp) and 94.8 % (1429 bp), respectively, and Desulfobulbus rhabdoformis (Lien et al., 1998) with sequence similarities of 94.0 (1453 bp) and 93.7 % (1457 bp), respectively. Sequence similarities with ‘Desulfobulbus marinus’ (Widdel & Pfennig, 1982) were 94.1 and 94.5 %, respectively, although the sequence length available for comparison was rather short (1211 bp).
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). The incomplete oxidation of propionate as an electron donor for sulfate-reduction and production of acetate and propionate from lactate and pyruvate without electron acceptors are both specific characteristics for Desulfobulbus species. Thus, strains Pro1T and Pro16 share several important features with species of the genus Desulfobulbus, including cell morphology and the composition of major respiratory quinone.
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). Strains Pro1T and Pro16 are the second group in the genus Desulfobulbus that have been reported to utilize carbohydrates as electron donors for sulfate reduction, although the range of carbohydrates utilized is rather limited compared with that of D. mediterraneus (Sass et al., 2002). D. mediterraneus is reported to ferment glucose and fructose in the absence of electron acceptors, whereas strain Pro1T only slightly consumed them and grew very weakly on them.
As shown in Table 1, the DNA G+C contents of Desulfobulbus species seem to be divided into two levels (47–50 % and about 59 %, respectively). The DNA G+C contents of strains Pro1T and Pro16 were almost consistent with those of the lower level. The CFA profiles of the novel strains are compared with those of the closest relatives, D. mediterraneus and D. rhabdoformis, in Table 2. Strains Pro1T and Pro16 shared a common profile of overall CFA composition with these phylogenetic relatives. Although differences in the cultivation conditions of cells should be considered when comparing CFA profiles, major differences between the species were found for CFAs such as C14 : 0, C15 : 0, C16 : 17, C17 : 16 and C18 : 17.
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Description of Desulfobulbus japonicus sp. nov.
Desulfobulbus japonicus (ja.po'ni.cus. N.L. masc. adj. japonicus pertaining to Japan, from where the type strain was originally isolated).
Cells are rod-shaped, 0.8–1.6 µm wide and 1.4–2.9 µm long. Strictly anaerobic. Gram-negative. Motile by a single polar flagellum. Non-spore-forming. Catalase and oxidase activities are not detected. Colonies are greyish and thin and spread on slant media. The NaCl concentration range for growth is 1.0–7.0 (w/v), with an optimum at 3.0 % (w/v). The temperature range for growth is 15–35 °C, with an optimum at 35 °C. The pH range for growth is 6.1–7.5, with an optimum at pH 6.7. Utilizes formate, propionate, pyruvate, lactate, fumarate, malate, ethanol, propanol, butanol, glycerol, alanine, glucose, fructose and H2 as electron donors for sulfate reduction. Does not use acetate, butyrate, succinate, methanol, glycine, serine, aspartate, glutamate, arabinose, ribose, xylose, galactose, mannose, rhamnose, sorbose, cellobiose, lactose, melibiose or sucrose. Almost all organic electron donors are incompletely oxidized to acetate. Sulfate and thiosulfate serve as electron acceptors. Does not use sulfite or fumarate. Pyruvate and lactate are fermented in the absence of electron acceptors to acetate and propionate. The genomic DNA G+C content is 46.0–48.6 mol%. Major cellular fatty acids are C15 : 0, C16 : 0, C17 : 16 and C18 : 17. The major respiratory quinone is menaquinone MK-5(H2).
The type strain, Pro1T (=JCM 14043T=DSM 18378T), was isolated from an estuarine sediment located on the side of the Sea of Japan of the Japanese islands. Strain Pro16 (=JCM 14044=DSM 18379) is a reference strain.
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ACKNOWLEDGEMENTS |
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