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1 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, D-38124 Braunschweig, Germany
2 Institute of Biochemistry and Physiology of Microorganisms, 142292 Pushchino, Moscow Region, Russia
3 Lehrstuhl für Mikrobiologie, Technische Universität Braunschweig, Germany
Correspondence
Hans Hippe
h.h.hippe{at}t-online.de
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ABSTRACT |
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The GenBank accession numbers for the 16S rRNA gene sequences described in this paper are: AJ237604, Desulfomicrobium macestii DSM 4194T; AJ277894, Desulfomicrobium baculatum DSM 4028T; AJ277897, Desulfomicrobium norvegicum DSM 1741T; AJ237606, Desulfobacula phenolica DSM 3384T; AJ237601, Desulfobacterium anilini DSM 4660T.
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was described for a motile, Gram-negative, rod-shaped, non-spore-forming, sulfate-reducing strain from the water of a sulfide spring at Matsesta, Russia. The strain used hydrogen, formate, lactate, pyruvate and ethanol for growth and sulfate as an electron donor, and did not contain desulfoviridin. On the basis of these phenotypic properties, this strain (M-9T) was classified as a member of the genus Desulfobacterium Widdel and Bak (1992). This report presents data that are inconsistent with the current classification, but provides evidence for reclassification of strain M-9T (=VKM B-1598T=DSM 4194T) as a member of the genus Desulfomicrobium.
The genus Desulfomicrobium was described by Rozanova et al. (1988, 1994) for rod-shaped, non-spore-forming, Gram-negative, sulfate-reducing bacteria that do not contain desulfoviridin and perform incomplete oxidation of organic compounds to acetate. At the time of writing, the genus includes the species Desulfomicrobium baculatum (Rozanova et al. 1988, 1994), Desulfomicrobium apsheronum (Rozanova et al., 1988, 1994), Desulfomicrobium escambiense (Sharak Genthner et al., 1994, 1996), Desulfomicrobium norvegicum (Sharak Genthner et al., 1997), Desulfomicrobium orale (Langendijk et al., 2001) and ‘Desulfomicrobium hypogeium’ (Krumholz et al., 1999); these have been isolated from various habitats, such as the stratal water of an oil deposit, manganese ore, subsurface sandstone, forest pond, brackish water, fresh water and marine water or sediment, respectively. Phylogenetically, the genus Desulfomicrobium groups within the family Desulfovibrionaceae (Devereux et al., 1990; Tourova et al., 1998) and is phylogenetically, physiologically and chemotaxonomically distinct (e.g. in its type of oxidation of organic compounds, DNA G+C content, cellular fatty acid pattern and menaquinones) from members of the genus Desulfobacterium (Collins & Widdel, 1986; Widdel & Bak, 1992).
Previously, Desulfobacterium macestii had been shown to grow in the absence of organic carbon sources and in the presence of sulfate with hydrogen or formate as a chemoautotroph, and with ethanol, lactate or pyruvate as a chemo-organotroph (Gogotova & Vainshtein, 1989). These results were confirmed in the present study. In addition, it was found that Desulfobacterium macestii was able to grow on n-butanol and 1,2-propanediol and weakly on n-propanol, but failed to grow on isobutanol, 1,4-butanediol or 2,3-butanediol. In contrast to previous results, fumarate and malate were used as carbon and electron sources in the presence of sulfate. In repeated experiments that used 10–15 mM organic substrate and 10 mM sulfate, cultures grew up to an optical density of 0·260–0·350 and produced 3–5 mM sulfide. In the absence of sulfate, fumarate and pyruvate were fermented, while lactate and malate were not. No growth occurred on lactate with nitrate as the electron acceptor.
Extraction of genomic DNA, PCR-mediated amplification of 16S rDNA and direct sequencing of the purified PCR product were carried out according to Rainey et al. (1996). The 16S rDNA sequences were aligned manually with published sequences obtained from GenBank/EMBL. Evolutionary distances were calculated by the method of Jukes & Cantor (1969). Phylogenetic dendrograms were reconstructed as described by DeSoete (1983). Bootstrap analysis was used to evaluate the neighbour-joining tree topology by performing 500 resamplings (Felsenstein, 1985).
Species of the genus Desulfomicrobium form three lineages: Desulfomicrobium orale is the deepest-branching member of the genus and shows <96 % 16S rRNA gene sequence similarity with the other species. The next-deepest-branching member is Desulfomicrobium escambiense, which shares between 98·1 and 98·3 % similarity with members of the third lineage; this lineage comprises the closely related species Desulfomicrobium baculatum, Desulfomicrobium norvegicum, Desulfomicrobium apsheronum and ‘Desulfomicrobium hypogeium’ (99·4–99·8 % similarity). This cluster also contains Desulfobacterium macestii DSM 4194T, which shares 100 % 16S rRNA gene similarity with Desulfomicrobium norvegicum DSM 1741T, 99·8 % similarity with Desulfomicrobium baculatum DSM 4028T and ‘Desulfomicrobium hypogeium’ CN-AT and 99·6 % similarity with Desulfomicrobium apsheronum DSM 5918T. The published sequence of Desulfomicrobium baculatum VKM B-1378T (GenBank no. AF030438) differs from the newly analysed sequence of Desulfomicrobium baculatum DSM 4028T (GenBank no. AJ277894) by 1·3 %. This in the sequence explains the different phylogenetic positions of Desulfomicrobium baculatum, which branches with Desulfomicrobium apsheronum in the study of Langendijk et al. (2001) and with Desulfomicrobium norvegicum and Desulfobacterium macestii in the present study (Fig. 1).
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. Cells were harvested at the end of the exponential-growth phase and washed twice with 1 % (w/v) NaCl. Saponification, methylation of fatty acids, extraction, separation by GC and analysis of the fatty acid methyl esters were done as described previously (Vainshtein et al., 1992; Meier et al., 1993), by using the MIDI system (Microbial ID). The overall fatty acid composition of Desulfobacterium macestii was very similar to those of species of the genus Desulfomicrobium (Vainshtein et al., 1992; Langendijk et al., 2001) but distinct from those of representatives of the genera Desulfovibrio and Desulfobacterium (Taylor & Parkes, 1983; Dowling et al., 1986; Vainshtein et al., 1992). The predominant fatty acids in Desulfobacterium macestii were the branched-chained, odd-numbered fatty acids iso C15 : 0 (13·6 %), iso C17 : 0 (10·1 %) and iso C17 : 1
10c (32·9 %), as well as significant amounts of the unbranched, even-numbered fatty acids C16 : 0 (4·2 %), C18 : 0 (5·5 %) and C18 : 17c (6·3 %). In particular, iso C17 : 110c has been regarded as a marker for members of the Desulfovibrionaceae (Edlund et al., 1985; Taylor & Parkes, 1985; Tourova et al., 1998). 10-Methyl fatty acids, claimed to be characteristic of the genera Desulfobacter and Desulfobacterium (Dowling et al.,1986; Vainshtein et al., 1992), were absent from Desulfobacterium macestii, as well as from members of the genera Desulfomicrobium and Desulfovibrio. Desulfobacterium macestii differs only slightly from the phylogenetically closely related type strains of Desulfomicrobium norvegicum and Desulfomicrobium baculatum in quantitative, but not qualitative, fatty acid composition.
Cells for isoprenoid quinone analysis (Kroppenstedt, 1985; Hippe et al., 1997) were grown in the medium described above, modified to contain 10 g NaCl l-1 and 2·2 g Na-lactate l-1 to replace pyruvate and malate. The principal isoprenoid quinone in Desulfobacterium macestii was menaquinone with six isoprenoid units (MK-6). This is not in accordance with the menaquinone composition of Desulfobacterium species, which contain either MK-7 or MK-7(H2) as the major menaquinone (Collins & Widdel, 1986; Widdel & Bak, 1992). MK-6 is typically found in members of the family Desulfovibrionaceae and has also been detected in Desulfomicrobium norvegicum (formerly Desulfovibrio desulfuricans Norway 4) (Collins & Widdel, 1986).
Genomic similarities between the closely related species Desulfomicrobium norvegicum DSM 1741T and Desulfobacterium macestii DSM 4194T were analysed by DNA–DNA reassociation, following the renaturation method (Escara & Hutton, 1980; Huß et al., 1983; Jahnke, 1992) in 1x SSC (saline/sodium citrate) buffer plus 10 % DMSO at 65 °C. The mean binary reassociation value of triple determination was 71·7 % (range, 69·7–73·5 %), which is close to the recommended threshold value for species delineation (70 %; Wayne et al., 1987). The reassociation value for Desulfomicrobium baculatum DSM 4028T and Desulfomicrobium norvegicum ATCC 27774T is only 27·4 % (Sharak Genthner et al., 1997), indicating that strain DSM 4028T and Desulfobacterium macestii DSM 4194T also share significantly less than 70 % similarity.
Results of phylogenetic and chemotaxonomic studies clearly identify Desulfobacterium macestii as a member of the genus Desulfomicrobium within the family Desulfovibrionaceae. DNA–DNA hybridization points toward a close relationship with Desulfomicrobium norvegicum, but Desulfobacterium macestii DSM 4194T differs from the type strain of Desulfomicrobium norvegicum, as well as from Desulfomicrobium baculatum, by chemoautotrophic growth on H2 plus CO2, lack of growth on malate in the absence of sulfate, and sulfate reduction during growth on fumarate and malate in the presence of sulfate. We therefore propose to reclassify Desulfobacterium macestii Gogotova and Vainshtein (1989) as Desulfomicrobium macestii comb. nov.
Description of Desulfomicrobium macestii comb. nov.
Desulfomicrobium macestii (ma. ces'ti.i. L. neut. adj. macestii referring to the town Matsesta at Sotschi, Black Sea, Russia, from where the type strain was isolated).
The description is based on the data of Gogotova & Vainshtein (1989) and data from recent studies.
Cells are straight rods, 0·7x1·9–2·0 µm in size and motile by a single polar flagellum. Spores are not formed. Gram-negative. Strictly anaerobic chemo-organotroph or chemoautotroph. H2, formate, pyruvate, lactate, ethanol, propanol, butanol, 1,2-propanediol, fumarate and malate are used as electron donors. Incomplete oxidation. Acetate, butyrate, methanol, isobutanol, 1,4-butanediol, 2,3-butanediol, choline, glucose and sucrose are not utilized. Sulfate, sulfite and thiosulfate serve as electron acceptors and are reduced to H2S. Fermentative growth occurs on pyruvate and fumarate in the absence of sulfate. Optimum temperature for growth is 35 °C, range is 15–40 °C; optimum pH for growth is 7·2, range is 6·5–8·0. Optimum growth occurs in 1·3 % NaCl, range 0–2·5 %. Major fatty acids are branched-chain and odd-numbered: iso C15 : 0, iso C17 : 0 and iso C17 : 110c. Principal menaquinone is MK-6. Cells contain b- and c-type cytochromes and an active hydrogenase. Desulfoviridin is not present. DNA base ratio is 58·0 mol% G+C (by thermal denaturation).
The type strain is M-9T (=VKM B-1598T =DSM 4194T). Isolated from a sulfide spring at Matsesta, Russia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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