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1 Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia
2 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia
3 Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
Correspondence
Svetlana N. Dedysh
s.dedysh{at}mtu-net.ru
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7c. The DNA G+C content of strain T4T is 63·3 mol%. The three strains possess almost identical 16S rRNA gene sequences and are most closely related to two previously identified species of Methylocella, Methylocella palustris (97 % similarity) and Methylocella silvestris (97·5 % similarity). DNA–DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36 %. Thus, the tundra strains represent a novel species, for which the name Methylocella tundrae sp. nov. is proposed. Strain T4T (=DSM 15673T=NCIMB 13949T) is the type strain.
Published online ahead of print on 25 July 2003 as DOI 10.1099/ijs.0.02805-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and partial sequences of the mmoX and mxaF genes of Methylocella tundrae strain T4T are respectively AJ555244, AJ555245 and AJ555246.
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- and 
-Proteobacteria. Only two of these 11 genera, Methylocella and Methylocapsa, are represented by acidophilic organisms (Dedysh et al., 2000
, 2002). These bacteria eluded isolation for a long time, but were obtained in culture using new enrichment media with low dissolved salt content (Dedysh et al., 1998a, b). Several characteristics of the genus Methylocella make it unique compared with other methanotrophic bacteria. Representatives of this genus do not contain an intracytoplasmic membrane (ICM) system like that in all other known methanotrophs and appear to possess only a soluble form of methane monooxygenase (sMMO). The first isolates of this genus were obtained from boreal Sphagnum peat bogs and described as Methylocella palustris. Later, another species, Methylocella silvestris, was isolated from an acidic forest soil (Dunfield et al., 2003). This suggests that Methylocella are widespread geographically, in acidic terrestrial soils as well as in wetlands. The very recent enrichment of bacteria morphologically similar to Methylocella from tundra wetlands has shown that distribution of these methanotrophs may not be limited to ecosystems of the temperate zone (Berestovskaya et al., 2002). In this report, three strains of the genus Methylocella, obtained from tundra peat enrichments, are described and a novel species, Methylocella tundrae sp. nov., is proposed.
Strains T4T, TCh1 and TY1 were isolated from acidophilic, methanotrophic enrichments obtained from Sphagnum peat of three tundra wetland sites in northern Russia, Vorkuta, Chukotka and Yugorsk (Berestovskaya et al., 2002). Cell suspensions of these enrichments were spread-plated onto the surface of agar medium M2, pH 5·5 (Dedysh et al., 1998a), and the plates were incubated at 15 °C in desiccators under a methane/air (30 : 70) gas mixture. The same medium (both agar and liquid) was used for further isolation and purification of methanotrophs. For growth in liquid medium, 500 ml screw-cap serum bottles were used with a headspace/liquid space ratio of 4 : 1. After inoculation, the bottles were sealed with silicone rubber septa and methane was added aseptically using a syringe equipped with a disposable filter (0·22 µm) to achieve a 15–20 % mixing ratio in the headspace. Bottles were incubated on a rotary shaker (120 r.p.m.) at 20 °C. Both phase-contrast and electron microscopy were used to check the methanotrophic isolates for contamination throughout the various stages of growth. The absence of heterotrophic satellites was checked by plating isolates on several media containing different organic substrates, as described previously (Dedysh et al., 2000). Morphological observations, examination of thin sections, tests for utilization of different carbon and nitrogen sources and enzyme assays were performed as described for Methylocella palustris and Methylocella silvestris (Dedysh et al., 2000; Dunfield et al., 2003). Growth of isolates was monitored by nephelometry at 410 nm for 2 weeks with methane as the sole growth substrate under a variety of conditions, including temperatures of 4–37 °C, pH 3·9–8·0 and NaCl concentrations of 0·01–2·00 % (w/v) using liquid cultures. For fatty acid analyses, cells were grown on liquid mineral medium M2 with methane and harvested in the late exponential growth phase. Analyses were performed by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). The SDS-based method used for extraction of genomic DNA from isolates has been described previously (Dedysh et al., 1998a). The DNA base composition of strain T4T was determined by thermal denaturation using a Unicam SP1800 spectrophotometer at a heating rate of 0·5 °C min-1. The G+C content was calculated from the equation of Owen et al. (1969) [G+C content (mol%)=(Tmx2·08)-106·4]. DNA of Escherichia coli K-12 was used as the standard. DNA–DNA hybridization of strain T4T and the type strains of the two known species of Methylocella, Methylocella palustris KT and Methylocella silvestris BL2T, was done on nitrocellulose membrane filters (Hybond-N) according to Lysenko et al. (1988). PCR-mediated amplification of the 16S rRNA gene from positions 28 to 1491 (numbering according to the International Union of Biochemistry nomenclature for Escherichia coli 16S rRNA) was carried out as described for Methylocella palustris KT (Dedysh et al., 1998b, 2000). A partial fragment of the mmoX gene encoding the -subunit of sMMO hydroxylase was amplified using primers and PCR conditions described by Auman et al. (2000) and a partial fragment of the mxaF gene, which encodes the large subunit of methanol dehydrogenase, was amplified using an approach developed by McDonald & Murrell (1997). Sequencing was performed as described previously (Dedysh et al., 2000) and phylogenetic analysis was carried out using the ARB program package (Strunk & Ludwig, 1996).
Two distinct types of colonies developed on M2 agar medium after 4 weeks of incubation. One type was represented by highly raised, semi-transparent, circular and slimy colonies that were composed of long polymorphic rods of bipolar appearance. These bacteria were further identified as Methylocella palustris (99·7–100·0 % 16S rRNA gene sequence similarity to Methylocella palustris KT). Colonies of the second type were less raised, not slimy, circular, opaque/cream-coloured and 1–3 mm in diameter. Liquid cultures of these bacteria displayed white turbidity and a surface pellicle was not formed. Three strains (T4T, TCh1 and TY1) were selected, each representing one of the three methanotrophic enrichments used for isolation, and the purity of these cultures was confirmed. Cells of these three strains were Gram-negative, non-motile, short, slightly curved rods or ovoids that occurred singly (Fig. 1a). The cells were 0·6–0·8 µm wide by 1·0–1·5 µm long and reproduced by normal cell division. Old cultures contained a large number of cells that were phase light in the middle and phase dark on both edges (Fig. 1b). In contrast to Methylocella palustris and Methylocella silvestris, cells of the tundra strains did not possess a macrocapsule and, thus, did not form slimy flakes or aggregates in liquid culture.
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). These cell-wall invaginations were less pronounced in methanol-grown bacteria. The cells possessed a microcapsule, as revealed in preparations prefixed with ruthenium red (Luft, 1964) (Fig. 1d). Thin sections were prepared from cells grown in medium with a low Cu2+ concentration (0·1 µm) (Fig. 1d, e) and cells grown in copper-sufficient medium (1 µm) (Fig. 1c). However, regardless of the copper content of the medium, an extensive ICM system typical of type I and type II methanotrophs was absent from cells of the tundra strains. As with other representatives of the genus Methylocella, cells of the novel isolates had an ICM composed of singular flattened or ovoid membrane vesicles located on the periphery of the cytoplasm (Fig. 1c, e). Both types of vesicle (flattened and ovoid) were bounded by three-layered membranes and each contained a homogeneous matrix of lower electron density with respect to the cytoplasm. Large granules of poly-
-hydroxybutyrate were observed in both methane- and methanol-grown cells. Strains T4T, TCh1 and TY1 were capable of growth on methane or methanol as sole carbon and energy sources. Slow growth was also observed on methylamine and ammonium formate. Similar to Methylocella silvestris, the isolates from tundra wetlands utilized methanol at a wide range of concentrations (0·01–2·00 %). The most active growth occurred at methanol concentrations of 0·5–1·0 % (v/v). Growth factors were not required. All three strains utilized ammonium salts, nitrates and yeast extract as nitrogen sources. Weak growth also occurred when L-alanine, L-serine, L-arginine, L-asparagine, L-glutamine or L-proline (0·05 %, w/v) were provided as nitrogen sources. Strains T4T, TCh1, and TY1 were capable of slow growth in liquid nitrogen-free medium; however, they grew better if methanol instead of methane was provided as a carbon source. Acetylene reduction activity of these bacteria was detectable only in microaerobic conditions (1·0–2·5 % O2 in flask headspace) and ranged from 0·1 to 0·4 nmol C2H4 (mg dry biomass)-1 h-1.
The isolates grew in the pH range 4·2–7·5 (optimum at pH 5·5–6·0). The temperature range for growth was 5–30 °C (optimum at 15 °C). No growth occurred at 37 °C. The culture generation time under methane (15 %, v/v), calculated from increases in OD410 in the exponential phase (5 days) of growth, was 18–45 h. Growth inhibition of 80 % was observed in the presence of NaCl in the medium at concentrations of 0·8–1·2 % (w/v), whereas NaCl at concentrations above 1·2 % completely inhibited growth.
Similar to all other currently known acidophilic methanotrophs, the major component of the phospholipid fatty acid (PLFA) profiles of the novel isolates was 11-cis-octadecenoic acid (18 : 17c), which comprised 60–62 % of the total PLFAs (Table 1). The PLFA component 10-cis-octadecenoic acid (18 : 18c), which is highly characteristic of the Methylosinus/Methylocystis group, was not found in strain T4T, TCh1 or TY1. The distinctive feature of the PLFA profiles of the tundra isolates was the presence of 17 : 0 cyclo and 19 : 08c cyclo fatty acids, which were not detected in other acidophilic methanotrophs.
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; Dunfield et al., 2003) (Table 2). They oxidized methane through methanol, formaldehyde and formate into CO2. They were capable of oxidizing naphthalene, thus implying the presence of sMMO. High activities of both hydroxypyruvate reductase and serine-glyoxylate aminotransferase were revealed, indicating that strain T4T assimilates C1 compounds via the serine pathway (Table 2). No hexulose phosphate synthase or ribulose bisphosphate carboxylase/oxygenase activities were found in cell extracts of the tundra isolates. The presence of the complete tricarboxylic acid cycle may be suggested since 2-oxoglutarate dehydrogenase was detected. Both the glutamate cycle enzymes and glutamate dehydrogenase are involved in nitrogen assimilation. Similar to other methanotrophs, strain T4T contained pyrophosphate- but not ATP-dependent 6-phosphofructokinase. Relatively high activities of the pentose phosphate cycle enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were measured in cell extracts of this methanotroph. Also, the glyoxylate cycle enzyme isocitrate lyase was found.
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-Proteobacteria (Fig. 2). They were most closely related to the acidophilic methanotrophic bacteria Methylocella palustris KT (97 % sequence similarity based on comparison of 1390 nt) and Methylocella silvestris BL2T (97·5 %). The 16S rRNA gene sequence similarity values to Methylocapsa acidiphila B2T and Beijerinckia indica subsp. indica were respectively 95 and 96 %. The DNA G+C content of strain T4T was 63·3 mol%. DNA–DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36 %.
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). The partial mxaF gene sequences of strains T4T, TCh1 and TY1 exhibited the highest identity to the corresponding gene fragment of Methylocella palustris KT (91 % nucleotide sequence identity and 97 % deduced amino acid sequence identity). Finally, pmoA genes were not detected in DNA of the novel isolates in PCRs conducted with either primer set A189/A682 (Holmes et al., 1995) or A189/mb661r (Costello & Lidstrom, 1999).
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Emended description of the genus Methylocella Dedysh et al. 2000
Methylocella (Me.thyl.o.cel'la. N.L. n. methylum the methyl group; L. n. cella a cell; N.L. n. Methylocella methyl-using cell).
Gram-negative, polymorphic rods or ovoids, 0·6–1·0x1·0–2·5 µm. Produces large, highly refractile, intracellular poly--hydroxybutyrate granules. Reproduces by normal binary cell division. Cells occur singly or in irregularly shaped aggregates, but do not form rosettes. Non-motile. Cells are not lysed by 2 % SDS. Encapsulated. Cells lack an extensive ICM system typical of both type I and type II methanotrophic bacteria, but contain a vesicular membrane system composed of singular flattened or ovoid vesicles connected to the cytoplasmic membrane. Possesses sMMO. Temperature range for growth is 4–30 °C (optimum at 15–25 °C); no growth at 37 °C. Growth occurs between pH 4·2 and 7·5. Highly sensitive to salt stress; prefers diluted media with a low salt content. Utilizes C1 compounds via the serine pathway. Does not contain enzymes of the ribulose monophosphate and ribulose bisphosphate pathways. Tricarboxylic acid cycle is complete. Fixes atmospheric nitrogen via an oxygen-sensitive nitrogenase. The major PLFAs are 18 : 17c acids. The G+C content is 60–63 mol%. Phylogenetically related to but clearly distinct from the type-II methanotrophic genera Methylocystis and Methylosinus in the -Proteobacteria; more closely affiliated with the acidophilic methanotrophic bacterium Methylocapsa acidiphila and the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica. Isolated from acidic wetlands, particularly Sphagnum peat bogs, and acidic soils. The type species is Methylocella palustris.
Description of Methylocella tundrae sp. nov.
Methylocella tundrae (tun'drae. N.L. gen. fem. n. tundrae from the tundra, the northern zone of Eurasia and North America).
Description as for the genus plus the following traits. Cells grown on methane are curved ovoids. Old cultures contain many cells that look phase light in the middle and phase dark on both edges. Cells do not possess a macrocapsule and colonies are not slimy like those of Methylocella palustris or Methylocella silvestris. Liquid cultures display homogeneous turbidity. An ICM system is composed of ovoid membrane vesicles and of singular flattened membrane vesicles aligned parallel to the cytoplasmic membrane. Optimal growth occurs at 15 °C and at pH 5·5–6·0. Capable of slow growth at 5 °C and pH 4·2. Carbon sources include methane, methanol, methylamine and formate. Utilizes methanol in a wide range of concentrations from 0·01 to 2·00 % (v/v). NaCl inhibits growth at concentrations above 0·8 % (w/v). The distinctive feature of the PLFA profile is the presence of 19 : 08c cyclo fatty acids. The DNA G+C content of the type strain is 63·3 mol%.
The type strain, T4T (=DSM 15673T=NCIMB 13949T), was isolated from an acidic Sphagnum peatland in Vorkuta region, northern Russia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Berestovskaya, Y. Y., Vasil'eva, L. V., Chestnykh, O. V. & Zavarzin, G. A. (2002). Methanotrophs of the psychrophilic microbial community of Russian arctic tundra. Microbiology (English translation of Mikrobiologiya) 71, 538–544.
Costello, A. M. & Lidstrom, M. E. (1999). Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl Environ Microbiol 65, 5066–5074.
Dedysh, S. N., Panikov, N. S. & Tiedje, J. M. (1998a). Acidophilic methanotrophic communities from Sphagnum peat bogs. Appl Environ Microbiol 64, 922–929.
Dedysh, S. N., Panikov, N. S., Liesack, W., Großkopf, R., Zhou, J. & Tiedje, J. M. (1998b). Isolation of acidophilic methane-oxidizing bacteria from northern peat wetlands. Science 282, 281–284.
Dedysh, S. N., Liesack, W., Khmelenina, V. N., Suzina, N. E., Trotsenko, Y. A., Semrau, J. D., Bares, A. M., Panikov, N. S. & Tiedje, J. M. (2000). Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs. Int J Syst Evol Microbiol 50, 955–969.[Abstract]
Dedysh, S. N., Khmelenina, V. N., Suzina, N. E., Trotsenko, Y. A., Semrau, J. D., Liesack, W. & Tiedje, J. M. (2002). Methylocapsa acidiphila gen. nov., sp. nov., a novel methane-oxidizing and dinitrogen-fixing acidophilic bacterium from Sphagnum bog. Int J Syst Evol Microbiol 52, 251–261.[Abstract]
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Lysenko, A. M., Gal'chenko, V. F. & Chernykh, N. A. (1988). Taxonomic study of obligate methanotrophic bacteria using the DNA–DNA hybridization technique. Microbiology (English translation of Mikrobiologiya) 57, 653–658.
McDonald, I. R. & Murrell, J. C. (1997). The methanol dehydrogenase structural gene mxaF and its use as a functional gene probe for methanotrophs and methylotrophs. Appl Environ Microbiol 63, 3218–3224.[Abstract]
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