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Institute of Microbiology, Russian Academy of Sciences, Prospekt 60, let Oktyabrya 7/2, 117811 Moscow, Russia
Correspondence
Tatyana Sokolova
tatso{at}mail.ru
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ABSTRACT |
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CO2+H2. No organic substrates were used as energy sources. For lithotrophic growth on CO, 0·2 g acetate or yeast extract l–1 was required but did not support growth in the absence of CO. Growth was observed in the temperature range 37–68 °C, the optimum being 55 °C. The pH range for growth was 6·7–9·5, the optimum pH being 8·0. The generation time under optimal conditions was 1·3 h. The DNA G+C content was 45 mol%. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization by strain 2204T. Thus, isolate 2204T was found to be the first known moderately thermophilic and alkalitolerant H2-producing anaerobic carboxydotroph. The novel bacterium fell within the cluster of the family Peptococcaceae within the low-G+C-content Gram-positive bacteria, where it formed a separate branch. On the basis of morphological, physiological and phylogenetic features, strain 2204T should be assigned to a novel genus and species, for which the name Thermincola carboxydiphila gen. nov., sp. nov. is proposed. The type strain is strain 2204T (=DSM 17129T=VKM B-2283T=JCM 13258T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Thermincola carboxydiphila strain 2204T is AY603000.
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MAIN TEXT |
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CO2+H2 (
G0=–20 kJ): Carboxydothermus hydrogenoformans (Svetlichny et al., 1991
), Caldanaerobacter subterraneus subsp. pacificus (Sokolova et al., 2001; Fardeau et al., 2004), Carboxydocella thermautotrophica (Sokolova et al., 2002) and Thermosinus carboxydivorans (Sokolova et al., 2004a). Recently, a hyperthermophilic archaeon of the genus Thermococcus able to grow by the same reaction was isolated from deep-sea hot vents (Sokolova et al., 2004b). All these organisms are neutrophiles, growing in the pH range 6·5–7·8. To date, alkalitolerant, CO-utilizing, H2-producing, thermophilic anaerobes have not been reported. Here we describe a novel anaerobic, moderately thermophilic, moderately alkaliphilic, CO-oxidizing, H2-producing bacterium isolated from an alkaline hot spring of the Baikal Lake area. Samples of hot water, mud and cyanobacterial mats were taken from freshwater thermal springs of the Baikal Lake area; the pH of the water varied from 6·8 to 9·5, and the original temperatures were in the range 51–72 °C. The samples were taken anaerobically in tightly stoppered bottles and transported to the laboratory at ambient temperature.
Unless otherwise mentioned, the medium used for enrichments and cultures contained the following (g l–1): NH4Cl (1), MgCl2.2H2O (0·33), CaCl2.6H2O (0·1), KCl (0·33), KH2PO4 (0·5), 1 ml trace mineral solution (Kevbrin & Zavarzin, 1992), 1 ml vitamin solution (Wolin et al., 1963) and resazurin (0·001). NaHCO3 (0·5 g l–1), Na2CO3 (0·5 g l–1), Na2S.9H2O (1 g l–1) and sodium acetate (2 g l–1) were added after boiling and cooling of the medium under the flow of nitrogen, and the pH was adjusted to 8·5–9·0 with 5 M HCl. Samples (10 ml) of the medium were placed into 50 ml bottles and the headspaces were filled with 100 % CO at atmospheric pressure. The incubation temperature was 55 °C. Growth was determined using light microscopy (MBI-3 microscope; LOMO) and GLC detection (GLC-Chrom 5; Laboratorni P
istrozhe Praha) of CO utilization and gaseous growth-product formation (Sokolova et al., 2001). After incubation at 55 °C on the medium with CO, several samples produced significant microbial growth. In most cases, short oval rods were predominant. From the sample of mud and cyanobacterial mat from a hot spring on the bank of the Bolshaya River (Barguzin Reserve), an enrichment culture was obtained that grew at pH 9·0 by the utilization of CO and the production of equimolar quantities of H2 and CO2. After 7 days incubation, in the stationary growth phase, the gas pressure increased 1·5–2-fold and the pH of the medium changed to neutral. The enrichment was used for further purification. After a number of serial-dilution transfers, colonies were obtained in roll-tubes prepared in 15 ml Hungate tubes on the same medium solidified by 5 % agar, with CO in the gas phase. Round, white, semi-transparent colonies (up to 1 mm in diameter) developed after 5 days incubation at 55 °C. Well-separated colonies were transferred to the same liquid medium as that used for the enrichment. A pure culture was obtained and designated strain 2204T.
Electron microscopy (JEM-100C apparatus; JEOL) and light microscopy were carried out as described previously (Sokolova et al., 2002). The ability of the novel isolate to use diverse energy substrates was tested with the same liquid mineral medium (both in the presence of 0·5 g yeast extract l–1 and in its absence) containing possible substrates at 2 g l–1 (final concentration), with 100 % N2 as the gas phase. Possible electron acceptors were added at a concentration of 2 g l–1 (or 10 g l–1 in the case of elemental sulfur). Ferric iron hydromorphic oxide (90 mM) was added to the same medium devoid of Na2S.9H2O. The cell density was determined by direct cell counting using a phase-contrast microscope at a magnification of x675.
The influence of the following antibiotics (50 µg ml–1) on growth was tested on the medium with CO as the gas phase: penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline.
DNA was prepared as described by Marmur (1961). The DNA G+C content was determined by means of melting-point analysis (Marmur & Doty 1962), using Escherichia coli K-12 DNA as a reference.
16S rRNA gene amplification, sequencing and sequence analyses were done as described previously (Sokolova et al., 2002).
The cells of isolate 2204T were straight, thick rods with rounded ends, about 0·5 µm wide and 0·6–3·0 µm long. The cells were motile and single or in short chains of three to five cells; sometimes cells formed aggregates of up to 15 cells. Spores were never observed in cultures. Cultures were not transferable after 5 min heat treatment at 100 °C. Electron microscopy of whole cells showed the presence of one or two lateral flagella (Fig. 1a). Ultrathin sections revealed Gram-positive cell-wall structure and a globular S-layer (Fig. 1b). The cytoplasmic membrane was often invaginated (Fig. 1b).
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Isolate 2204T grew in an atmosphere of 100 % CO on medium containing 0·2 g yeast extract l–1 or 0·2 g sodium acetate l–1. CO oxidation was coupled with equimolar H2 and CO2 formation, like the CO oxidation of previously described anaerobic hydrogenogenic bacteria (Svetlichny et al., 1991; Sokolova et al., 2001, 2002). Neither methane formation nor acetate formation was detected during the growth of isolate 2204T on CO. Either yeast extract or sodium acetate (0·2 g l –1) was found to be required for growth. The generation time under optimal growth conditions (55 °C, pH 8·0) was 1·3 h–1. Strain 2204T was unable to grow organotrophically on peptone or yeast extract or on starch, cellulose, cellobiose, sucrose, maltose, ribose, xylose, lactose, glucose, galactose, fructose, mannitol, sorbitol, pyruvate, acetate, formate, lactate, succinate, methanol, ethanol or glycerol. No growth was observed on an H2/CO2 gas mixture (80 : 20), on H2 or CO with ferric iron or on H2 or lactate with fumarate in the usual liquid mineral medium supplemented with 0·2 g sodium acetate l–1 and 0·5 g yeast extract l–1. No growth was observed on peptone, yeast extract, sucrose, pyruvate, acetate, formate, lactate, succinate, methanol, ethanol or glycerol in the presence of elemental sulfur, sulfate, thiosulfate or ferric iron. Elemental sulfur, thiosulfate, sulfate and nitrate did not stimulate growth and were not reduced during growth on CO.
Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization.
The DNA G+C content of strain 2204T was 45·4±1 mol%. The 16S rRNA gene sequence (1444 nt, corresponding to positions 38–1485 of E. coli numbering) was determined for strain 2204T. Preliminary comparisons using BLAST (http://www.ncbi.nih.gov/BLAST/) performed with representatives of the domain Bacteria revealed that the novel isolate was a member of the Bacillus–Clostridium subphylum of Gram-positive bacteria but was not phylogenetically related to any named organism. The closest match (94 % similarity) was with an uncultured clone, SHA-15, from an anaerobic 1,2-dichloropropane-dechlorinating mixed culture (Schlötelburg et al., 2000). The closest organism of the species with validly published names (93 % similarity) was Pelotomaculum thermopropionicum (Imachi et al., 2000).
Several phylogenetic trees were constructed by changing the compositions of reference organisms belonging to the Clostridium group. Regions of alignment uncertainties due to the presence of long inserts in 16S rRNA gene sequences around positions 80, 1040, 1140 and 1440 (E. coli numbering) of some members of this group (Rainey et al., 1993; Slobodkin et al., 1999) were omitted from sequence analyses. According to the phylogenetic analysis, the novel strain 2204T fell within the radiation of the family Peptococcaceae. A final comparison of 1327 nt of 16S rRNA gene sequences of strain 2204T and 40 reference strains of the family Peptococcaceae was made and used for the reconstruction of a phylogenetic tree and the calculation of sequence similarity. In a phylogenetic tree constructed by using the neighbour-joining algorithm (Jukes & Cantor, 1969) (Fig. 2), strain 2204T was not clustered exactly with any genus or species of the family Peptococcaceae, including the CO-utilizing hydrogenogens Carboxydothermus hydrogenoformans and Thermosinus carboxydivorans. The additional trees constructed by using other treeing algorithms, including option ‘transversion only’ for thermophilic organisms, had the same topology (data not shown). The level of sequence similarity of strain 2204T was relatively low and almost equal for all reference strains (82·4–88·3 %). Strain 2204T formed a single cluster with maximal bootstrap support only with uncultured clone SHA-15 (Schlötelburg et al., 2000); the level of similarity of complete 16S rRNA gene sequences was 92·9 %, so this clone may represent a micro-organism phylogenetically related to strain 2204T.
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); however, strain 2204T could not grow autotrophically, requiring acetate or yeast extract. All previously described carboxydotrophic hydrogenogens are neutrophiles; strain 2204T, which grows at pH values in the range 6·7–9·5, is the first alkalitolerant representative of this group (Table 1). According to 16S rRNA gene analyses, strain 2204T forms a new separate line of descent within the low-G+C-content Gram-positive subdivision of the Bacteria and could not be assigned to any genus with a validly published name. Thus, we propose a novel genus for it, namely Thermincola, with Thermincola carboxydiphila as the type species.
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Cells are non-spore-forming rods. Cell wall is of the Gram-positive type. Anaerobic, thermophilic bacteria. Capable of growth by anaerobic CO oxidation, producing molecular hydrogen and CO2. Does not grow by fermentation of organic substrates. The type species is Thermincola carboxydiphila. Habitat: terrestrial hot springs.
Description of Thermincola carboxydiphila sp. nov.
Thermincola carboxydiphila (car.bo.xy.di.phi'la. N.L. neut. n. carboxydum carbon monoxide; Gr. adj. philos loving; N.L. fem. adj. carboxydiphila loving carbon monoxide).
Cells are straight, thick rods, with rounded ends, about 0·5 µm wide and 0·6–3·0 µm long. Motile due to one or two lateral flagella. Cell wall is of the Gram-positive type. Obligately anaerobic. Grows in the temperature range 37–68 °C, with an optimum at 55 °C. Alkalitolerant: pH range from 6·7 to 9·5, with an optimum at pH 8·0. Grows chemolithotrophically on CO. Utilizes CO as sole energy source, with equimolar formation of H2 and CO2 according to the equation CO+H2OCO2+H2. The presence of 200 mg yeast extract or acetate l–1 is required for growth. Elemental sulfur, thiosulfate, sulfate and nitrate do not stimulate growth and are not reduced during growth on CO. Does not grow organotrophically on peptone, yeast extract, starch, cellulose, cellobiose, sucrose, maltose, ribose, xylose, lactose, glucose, galactose, fructose, mannitol, sorbitol, pyruvate, acetate, formate, lactate, succinate, methanol, ethanol or glycerol. H2/CO2 gas mixture (80 : 20) does not support growth. Does not grow on H2 or CO with ferric iron. Does not grow on peptone, yeast extract, sucrose, pyruvate, acetate, formate, lactate, succinate, methanol, ethanol or glycerol, in the presence of elemental sulfur, sulfate, thiosulfate or ferric iron. Growth is completely inhibited by penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline. The DNA G+C content of the type strain is 45·4±1 mol%.
The type strain is strain 2204T (=DSM 17129T=VKM B-2283T=JCM 13258T), isolated from a hot spring of the Baikal Lake region.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Imachi, H., Sekiguchi, Y., Kamagata, Y., Ohashi, A. & Harada, H. (2000). Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol 66, 3608–3615.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kevbrin, V. V. & Zavarzin, G. A. (1992). The influence of sulfur compounds on the growth of halophilic homoacetic bacterium Acetohalobium arabaticum. Microbiology (English translation of Mikrobiologiia) 61, 812–817.
Marmur, J. (1961). A procedure for the isolation of desoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]
Rainey, F. A., Ward, N. L., Morgan, H. W., Toalster, R. & Stackebrandt, E. (1993). Phylogenetic analysis of anaerobic thermophilic bacteria: aid for their reclassification. J Bacteriol 175, 4772–4779.
Schlötelburg, C., von Wintzingerode, F., Hauck, R., Hegemann, W. & Göbel, U. B. (2000). Bacteria of an anaerobic 1,2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia. Int J Syst Evol Microbiol 50, 1505–1511.[Abstract]
Slobodkin, A. I., Tourova, T. P., Kuznetsov, B. B., Kostrikina, N. A., Chernyh, N. A. & Bonch-Osmolovskaya, E. A. (1999). Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium. Int J Syst Bacteriol 49, 1471–1478.
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]
Sokolova, T. G., Kostrikina, N. A., Chernyh, N. A., Tourova, T. P., Kolganova, T. V. & Bonch-Osmolovskaya, E. A. (2002). Carboxydocella thermautotrophica gen. nov., sp. nov., a novel anaerobic, CO-utilizing thermophile from a Kamchatkan hot spring. Int J Syst Evol Microbiol 52, 1961–1967.[Abstract]
Sokolova, T. G., Gonzalez, J. M., Kostrikina, N. A., Chernyh, N. A., Slepova, T. V., Bonch-Osmolovskaya, E. A. & Robb, F. T. (2004a). Thermosinus carboxydivorans gen. nov., sp. nov., a new anaerobic thermophilic, carbon-monoxide-oxidizing, hydrogenogenic bacterium from a hot pool of Yellowstone National Park. Int J Syst Evol Microbiol 54, 2353–2359.
Sokolova, T. G., Jeanthon, C., Kostrikina, N. A., Chernyh, N. A., Lebedinsky, A. V., Stackebrandt, E. & Bonch-Osmolovskaya, E. A. (2004b). The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Extremophiles 8, 317–323.[Medline]
Svetlichny, V. A., Sokolova, T. G., Gerhardt, M., Ringpfeil, M., Kostrikina, N. A. & Zavarzin, G. A. (1991). Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing thermophilic anaerobic bacterium from hydrothermal environments of Kunashir Island. Syst Appl Microbiol 14, 254–260.
Wolin, E. A., Wolin, M. J. & Wolfe, R. S. (1963). Formation of methane by bacterial extracts. J Biol Chem 238, 2882–2886.
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