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1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Prospect 60-letiya Oktyabrya 7/2, 117 312 Moscow, Russia
2 Department of Microbiology, University of Georgia, Athens, GA 30602, USA
Correspondence
A. I. Slobodkin
aslobodkin{at}hotmail.com
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). This micro-organism grew via utilization of organic substrates or molecular hydrogen and, besides ferric iron, reduced several other electron acceptors. 16S rRNA gene sequence analysis placed T. ferrireducens within the Bacillus–Clostridium subphylum, with the most closely related micro-organism at the time of description being Ammonifex degensii (83 % similarity). The combination of physiological properties and phylogenetic position allowed the organism to be assigned to a new genus.
The genus Carboxydothermus contains a single species, Carboxydothermus hydrogenoformans, an anaerobic carbon monoxide-utilizing thermophilic bacterium (Svetlichny et al., 1991a). The name of this species was subsequently validly published (Svetlichny et al., 1991b). A partial 16S rRNA gene sequence of C. hydrogenoformans Z-2901T became available in 2000 (GenBank accession no. AF244579), and analysis revealed 98.4 % similarity between this sequence and that of T. ferrireducens JW/AS-Y7T (GenBank accession no. U76363) (Sokolova et al., 2001). The phylogenetic position of C. hydrogenoformans is confirmed by recently published whole-genome sequence data (Wu et al., 2005; GenBank accession no. NC_007503).
This phylogenetic resemblance motivated Henstra & Stams (2004) to investigate the physiological properties of both micro-organisms in more detail. They found that the growth substrates for C. hydrogenoformans are not restricted to CO and pyruvate, as was previously reported (Pusheva & Sokolova, 1995), but that this bacterium is able to utilize various electron donors and acceptors for growth. The range of compounds utilized was identical to those used by T. ferrireducens. On the other hand, T. ferrireducens was capable of growth on CO with 9,10-anthraquinone 2,6-disulfonate or fumarate as electron acceptors (Henstra & Stams, 2004). Thus, the similarities in phylogeny and metabolic properties between C. hydrogenoformans and T. ferrireducens warranted a clarification of their taxonomic positions, as given below.
C. hydrogenoformans DSM 6008T (=Z-2901T) and T. ferrireducens DSM 11255T (=JW/AS-Y7T) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and routinely cultivated at 65 °C in mineral media as described previously (Svetlichny et al., 1991a; Slobodkin et al., 1997) with 100 % CO as the headspace gas (C. hydrogenoformans) and with the addition of glycerol (30 mM) and fumarate (20 mM) (T. ferrireducens). Growth of both micro-organisms in the presence of Fe(III) was investigated in anaerobic bicarbonate-buffered medium (Slobodkin et al., 1997) supplied with 0.2 g yeast extract l–1 (Sigma). Ferrihydrite [poorly crystalline Fe(III) oxide] (90 mM), Fe(III) citrate (20 mM) or Fe(III) EDTA (15 mM) was provided as an electron acceptor and CO (100 % as headspace gas), H2 (in the presence of CO2 at a ratio of 80 : 20), formate (20 mM), lactate (20 mM) or glycerol (30 mM) was provided as an electron donor. In all experiments with different forms of Fe(III), the medium did not contain any reducing agents. Microscopy, cell counts and analysis of CO, acetate and Fe(II) were carried out as described elsewhere (Slobodkin et al., 1997; Sokolova et al., 2001). The DNA was extracted and purified by the method of Marmur (1961). DNA–DNA hybridization studies were performed by the optical reassociation method as described previously (Krivenko et al., 1990).
The important physiological difference between C. hydrogenoformans and T. ferrireducens in CO metabolism is H2 production by C. hydrogenoformans irrespective of the cultivation conditions. T. ferrireducens does not produce molecular hydrogen during CO utilization and is unable to grow on CO without an external electron acceptor. Growth of C. hydrogenoformans with Fe(III) and the ability of T. ferrireducens to utilize CO with Fe(III) as an electron acceptor have not been investigated previously. Our attempts to obtain sustainable growth of C. hydrogenoformans with either CO, glycerol or lactate as an electron donor and either ferrihydrite, Fe(III) citrate or Fe(III) EDTA as an electron acceptor did not yield positive results. The only electron donor/acceptor couple that supported growth and Fe(III) reduction by this organism was H2/ferrihydrite. Under these conditions, within 7 days of cultivation, the maximal cell yield was 3.5–4.0x107 cells ml–1, ferrihydrite was converted to a black magnetic precipitate with a Fe(II) content of 20–22 mM and no accumulation of acetate was found in the cultivation medium. Thus, the type species of the genus Carboxydothermus is capable of Fe(III) reduction. The inability of C. hydrogenoformans to grow on CO with Fe(III) is probably caused by the higher redox potential of the medium in comparison with the medium pre-reduced with Na2S.9H2O used in previous studies (Svetlichny et al., 1991a; Henstra & Stams, 2004). As evident from growth on H2/ferrihydrite, the hydrogenase of C. hydrogenoformans can work at higher redox potential than CO dehydrogenase. In contrast to C. hydrogenoformans, T. ferrireducens grew and utilized CO with ferrihydrite as an electron acceptor. After 7 days of cultivation, the maximal cell yield was 2.5–3.0x107 cells ml–1, ferrihydrite was converted to a black magnetic precipitate with a Fe(II) content of 26–28 mM and 14 mM CO was consumed from the gas phase (45 % of the initial concentration) without producing acetate.
Subsequently, DNA–DNA hybridization of T. ferrireducens DSM 11255T with C. hydrogenoformans DSM 6008T revealed a reassociation value of 53 %. This level of relatedness is characteristic of different species belonging to the same genus (Johnson, 1984).
Considering the physiological similarities, phylogenetic position and level of DNA–DNA relatedness, T. ferrireducens and C. hydrogenoformans should be members of the same genus. According to the Bacteriological Code (Lapage et al., 1992), the name Carboxydothermus has priority, thus T. ferrireducens has to be reclassified as a member of the genus Carboxydothermus. However, according to the original description (Svetlichny et al., 1991a), representatives of the genus Carboxydothermus utilize CO with equimolar formation of H2 and CO2, do not utilize other organic and inorganic substrates and do not reduce sulfur. Therefore, taking into account the data obtained by Henstra & Stams (2004) and those generated in this study, the description of the genus Carboxydothermus needs to be emended.
Emended description of the genus Carboxydothermus Svetlichny et al. 1991
Rod-shaped, Gram-type positive (eu)bacteria. Anaerobic and thermophilic. Neutrophilic. Utilize CO either with or without production of molecular hydrogen. Able to couple the reduction of external electron acceptors such as Fe(III), sulfite, thiosulfate, elemental sulfur, nitrate, fumarate or 9,10-anthraquinone 2,6-disulfonate with oxidation of organic acids, polyols and H2. In the presence as well as in the absence of an electron acceptor, organic substrates are oxidized incompletely to acetate as the main metabolic product. Do not reduce sulfate.
Description of Carboxydothermus ferrireducens comb. nov.
Carboxydothermus ferrireducens [fer.ri.re.du'cens. L. n. ferrum iron; L. part. adj. reducens converting to a different state; N.L. part. adj. ferrireducens reducing (ferric) iron].
Basonym: Thermoterrabacterium ferrireducens Slobodkin et al. 1997.
The description is based on that published previously for Thermoterrabacterium ferrireducens (Slobodkin et al., 1997) with the additional data obtained by Gavrilov et al. (2003), Henstra & Stams (2004) and during this study. Cells are straight to slightly curved rods, 0.3–0.4 µm in diameter and 1.6–2.7 µm in length with rounded ends. Cells stain Gram-positive. Cells occur singly or in pairs, with retarded peritrichous flagellation, and exhibit tumbling motility. Spores are not observed. Anaerobic. Grows in mineral medium without supplementation with complex media components. The temperature range for growth is 50–74 °C with an optimum at 65 °C. The pH65 °C range for growth is 5.5–7.6, with an optimum at 6.0–6.2. No growth was observed at or below 48 °C, at or above 76 °C, at or below pH65 °C 5.3 and at or above pH65 °C 7.8. Growth occurs at NaCl concentrations in the range of 0–1.0 % (w/v). In the presence of CO2, the type strain is able to grow with glycerol as the only organic carbon source. Couples the oxidation of glycerol to reduction of ferrihydrite [poorly crystalline Fe(III) oxide], Fe(III) citrate or Fe(III) EDTA as electron acceptors. In the presence as well as in the absence of Fe(III) and in the presence of CO2, glycerol is oxidized incompletely to acetate as the only organic metabolic product, leading to a ratio of more than one acetate molecule produced per glycerol molecule consumed. Does not produce H2. Utilizes glycerol, lactate, 1,2-propanediol, glycerate, pyruvate, yeast extract and peptone. Grows weakly on glucose, fructose and mannose, producing acetate as the only organic product. In the presence of ferrihydrite, grows lithoautotrophically with molecular hydrogen as an electron donor and CO2 as the only carbon source. Utilizes CO with ferrihydrite, fumarate or 9,10-anthraquinone 2,6-disulfonate as electron acceptors, without the production of molecular hydrogen or acetate. No growth occurs with acetate, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, propionate, acetone, ethylene glycol, 1,3-propanediol, fumarate, succinate, phenol, benzoate, 9,10-anthraquinone 2,6-disulfonate, starch, olive oil, elemental sulfur, sucrose, galactose, xylose, cellobiose or arabinose with or without Fe(III). Reduces Fe(III) to Fe(II), 9,10-anthraquinone 2,6-disulfonate to 9,10-anthrahydroquinone 2,6-disulfonate, fumarate to succinate, thiosulfate to elemental sulfur or sulfide, sulfite to sulfide, elemental sulfur to sulfide and nitrate to nitrite and ammonium. Does not reduce MnO2 or sulfate. Growth is inhibited by chloramphenicol, erythromycin and rifampicin at 100 µg ml–1 but not by 100 µg ampicillin, streptomycin or tetracycline ml–1. The DNA base composition is 41 mol% G+C (HPLC).
The type strain is JW/AS-Y7T (=DSM 11255T=VKM B-2392T), isolated from a freshwater hot spring in the Calcite Springs area of Yellowstone National Park, WY, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Henstra, A. M. & Stams, A. J. M. (2004). Novel physiological features of Carboxydothermus hydrogenoformans and Thermoterrabacterium ferrireducens. Appl Environ Microbiol 70, 7236–7240.
Johnson, J. L. (1984). Nucleic acids in bacterial classification. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 8–11. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Krivenko, V. V., Vadachkoriya, R. M., Chernykh, N. A., Mityushina, L. L. & Krassilnikova, E. N. (1990). Clostridium uzonii sp. nov., an anaerobic thermophilic glycolytic bacterium from the hot springs of Kamchatka. Microbiology (English translation of Mikrobiologiia) 59, 741–749.
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seeliger, H. P. R. & Clark, W. A. (editors) (1992). International Code of Nomenclature of Bacteria (1990 Revision). Bacteriological Code. Washington, DC: American Society for Microbiology.
Marmur, J. (1961). A procedure for the isolation of desoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Pusheva, M. A. & Sokolova, T. G. (1995). Distribution of CO-dehydrogenase activity in anaerobic thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans grown at the expense of CO or pyruvate. Microbiology (English translation of Mikrobiologiia) 64, 491–495.
Slobodkin, A. I., Reysenbach, A.-L., Strutz, N., Dreier, M. & Wiegel, J. (1997). Thermoterrabacterium ferrireducens gen. nov., sp. nov. a thermophilic anaerobic, dissimilatory Fe(III)-reducing bacterium from a continental hot spring. Int J Syst Bacteriol 47, 541–547.
Sokolova, T. G., Gonzalez, J. M., Kostrikina, N. A., Chernyh, N. A., Tourova, T. P., Kato, C., Bonch-Osmolovskaya, E. A. & Robb, F. T. (2001). Carboxydobrachium pacificum gen. nov., sp. nov., a new anaerobic, thermophilic, CO-utilizing marine bacterium from Okinawa Trough. Int J Syst Evol Microbiol 51, 141–149.[Abstract]
Svetlichny, V. A., Sokolova, T. G., Gerhardt, M., Ringpfeil, M., Kostrikina, N. A. & Zavarzin, G. A. (1991a). Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing thermophilic anaerobic bacterium from hydrothermal environments of Kunashir Island. Syst Appl Microbiol 14, 245–260.
Svetlichny, V. A., Sokolova, T. G., Gerhardt, M., Ringpfeil, M., Kostrikina, N. A. & Zavarzin, G. A. (1991b). Carboxydothermus hydrogenoformans gen. nov., sp. nov. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB, List no. 39. Int J Syst Bacteriol 41, 580–581.
Wu, M., Ren, Q., Durkin, A. S. & 14 other authors (2005). Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901. PLoS Genet 1, e65.[CrossRef][Medline]
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