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1 Energy and Technology Laboratories, Osaka Gas Co., Ltd, 6-19-9 Torishima Konohana-ku, Osaka 554-0051, Japan
2 G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia
Correspondence
Yuri A. Trotsenko
trotsenko{at}ibpm.pushchino.ru
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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9c (35·2 %) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. On the basis of comparative phenotypic and genotypic characteristics, a new genus and species, Methylothermus thermalis gen. nov., sp. nov., is proposed, with MYHTT as the type strain (=VKM B-2345T=IPOD FERM P-19714T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and partial sequence of the pmoA gene of Methylothermus thermalis strain MYHTT are AY829009 and AY829010, respectively.
A figure showing the oxidation and assimilation of 14C-methane by cells of strain MYHTT is available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Nevertheless, in the past decade, investigations of extreme environments with high and low pH, temperatures or salinities have led to the discovery of a variety of extremophilic and extremotolerant methanotrophs (Murrell et al., 1998; Trotsenko & Khmelenina, 2002).
The solubility of CH4 and O2 in aqueous solution drops with increasing temperature, thus limiting growth of methanotrophic bacteria. However, gas solubility in natural waters of low ionic strength (<100 mM) decreases by only one-third when the temperature increases from 30 to 60 °C. This explains the existence of methanotrophs in various habitats with elevated temperatures. The earliest described heat-tolerant methanotroph was Methylococcus capsulatus, which grows at temperatures up to 50 °C (Foster & Davis, 1966; Whittenbury et al., 1970). Several other moderately thermophilic and thermotolerant species, including Methylococcus thermophilus, ‘Methylococcus ucrainicus’, Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile, were subsequently described (Malashenko et al., 1975; Malashenko, 1976; Bodrossy et al., 1995, 1997, 1999; Eshinimaev et al., 2004).
Very recently, a representative of a novel group of truly thermophilic methanotrophs, strain HB, was isolated from Hungarian and Japanese hot springs (Bodrossy et al., 1999), with temperature limits for growth between 40 and 70 °C and an optimum at 55–62 °C. Strain HB was closely related to the known thermotolerant Methylococcus and Methylocaldum species, as indicated by 16S rRNA and particulate methane monooxygenase (pmoA) gene sequence analyses. Strain HB represented a new genus, and the informal name ‘Methylothermus’ was proposed for this methanotroph (Bodrossy et al., 1999). However, no comparative studies of the phenotypic features of strain HB with those of known thermophilic and thermotolerant methanotrophs have been performed. Moreover, this organism is no longer extant (K. L. Kovács, personal communication), which prompted our attempts to isolate and characterize thermophilic methanotrophs from a Japanese hot spring. Herein, we describe a novel moderately thermophilic methanotrophic strain.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Growth conditions.
For the enrichment and isolation of methanotrophs, a basal ammonium nitrate mineral salt (ANMS) medium was used, containing (g l–1): KNO3, 0·25; NH4Cl, 0·25; KH2PO4, 0·13; Na2HPO4.12H2O, 0·358; MgSO4.7H2O, 0·4; CaCl2, 0·1; ferric-EDTA, 0·00019; and Na2MoO4.6H2O, 0·00013. To this medium was added 0·5 ml l–1 of a stock solution of trace elements, containing (g l–1): CuSO4.5H2O, 0·2; FeSO4.7H2O, 0·5; ZnSO4.7H2O, 0·4; H3BO3, 0·015; CoCl2.6H2O, 0·05; disodium EDTA, 0·25; MnCl2.4H2O, 0·02; and NiCl2.6H2O, 0·01. The pH of the medium was adjusted to 6·8 with NaOH. In addition, 2·5 ml l–1 of a filter-sterilized vitamin stock solution [mg l–1: thiamine hydrochloride, 10; nicotinic acid, 20; pyridoxamine, 10; p-aminobenzoic acid, 10; riboflavin, 20; biotin, 1; and cyanocobalamin (vitamin B12), 1] was added to media used for the enrichment, which was performed at 59 °C in 50 ml ANMS medium in 250 ml conical flasks. The gas phase of the flasks was replaced by a methane/air mixture (1 : 1) every 3–4 days. The cultures were shaken at 100 r.p.m. (Clim-O-Shaker).
Isolation of methanotrophs.
The mineral composition of the growth medium was optimized, based on an analysis of the methane consumption activities. Rates of 14C-methane oxidation and assimilation by cells incubated in basal ANMS medium and in two- or fourfold-concentrated media, and also in ANMS medium containing various concentrations of trace elements, were estimated by using the method described previously (Sokolov & Trotsenko, 1995). Since the rate of methane oxidation was higher at elevated concentrations of the trace elements (two- or fourfold) and both processes (methane oxidation and methane assimilation) were inhibited by CaCl2, the medium was modified slightly and then used for pure culture isolation. The modified (M) medium contained (g l–1): NH4Cl, 0·5; KH2PO4, 0·13; Na2HPO4.12H2O, 0·358; MgSO4.7H2O, 0·5; CaCl2, 0·02; ferric-EDTA, 0·0038; Na2MoO4.6H2O, 0·00026; and 1 ml l–1 of a stock solution of trace elements containing (g l–1): CuSO4.5H2O, 0·2; FeSO4.7H2O, 0·5; ZnSO4.7H2O, 0·4; H3BO3, 0·015; CoCl2.6H2O, 0·05; disodium EDTA, 0·25; MnCl2.4H2O, 0·02; and NiCl2.6H2O, 0·01. M medium was not supplemented with vitamin solution.
After two or three passages of the enrichment in liquid ANMS and then in M medium, serial dilutions were spread onto plates containing a mixture of 80 % M medium and 20 % autoclaved culture fluid from the initial enrichment, and 2·0 % (w/v) Bacto agar (Difco). The plates were incubated for 1–2 weeks at 53 °C in anaerobic jars filled with a methane/air (1 : 1) gas mixture. Individual colonies were restreaked onto fresh plates and reincubated. The resulting single colonies were checked for purity by light microscopy and placed into liquid M medium. Pure cultures were routinely maintained at 53 °C on solidified M medium. Absence of growth on various organic solid media and cell uniformity tested by light and electron microscopy were used as criteria for culture purity.
Electron microscopy.
Ultrathin sections of exponentially grown cells and negatively stained preparations were prepared and examined using a JEOL JEM 100B electron microscope, as described previously (Khmelenina et al., 1999).
Utilizable carbon and nitrogen sources.
The ability of the isolate to use various organic compounds as a carbon source was tested in liquid M medium supplemented with autoclaved or filter-sterilized substrates (methylamine, formate, formaldehyde, formamide, acetate, pyruvate, citrate, malate, succinate, D-glucose, D-xylose, D-arabinose, maltose, sucrose, mannitol, ethanol, glycerol and yeast extract) at a final concentration of 0·5 g l–1. The ability of the isolate to grow on methanol was tested using a liquid mineral medium containing 0·05–1 % (v/v) methanol. Nitrogen sources were tested by using agar medium in which NH4Cl was replaced by one of the following compounds at a final concentration of 0·05 % (w/v): KNO3, NaNO2, (NH4)2SO4, methylamine, dimethylamine, urea, formamide, glycine, L-alanine, L-lysine, L-arginine, L-glutamate, L-glutamine, L-asparagine, L-lysine, L-aspartate, L-tryptophan, L-methionine, L-threonine, L-cysteine, L-histidine, Tris, disodium EDTA, Casamino acids and yeast extract.
Effect of pH, temperature and salinity on growth.
The temperature range for growth was estimated by growing the isolate in liquid M medium at 26, 30, 37, 54, 57, 59, 62, 65, 67 and 69 °C. The effect of pH was investigated at an optimum temperature of 57 °C, in M medium supplemented with NaH2PO4/Na2HPO4 buffer of various pH values (4·2–8·5), at a final concentration of 0·025 M. To test the salt-dependence of growth, NaCl was added to M medium at 0·25, 0·5, 1, 2 or 3 % (w/v); 10 ml of the culture grown at pH 6·8 was used for inoculation. Aliquots were taken at 12–18 h intervals and the optical density at 600 nm (OD600) was measured. The specific growth rate was calculated from increases in OD600 during the exponential growth phase. To test the copper-dependence of 14CH4 assimilation and oxidation rates, the cells were suspended in ANMS medium containing 0·4 µM CuSO4.5H2O, and additionally supplemented with CuSO4.5H2O (0, 0·5, 1, 2 and 4 µM). The cell suspensions were incubated at 57 °C and the radioactivity incorporated into the biomass or carbon dioxide was measured as described below.
Antibiotic sensitivity.
To test for antibiotic sensitivity, the cells were spread onto plates of solidified M medium with discs (Difco) containing the following antibiotics (µg ml–1): neomycin, 30; kanamycin, 30; ampicillin, 10; erythromycin, 15; lincomycin, 2; gentamicin, 10; novobiocin, 30; nalidixic acid, 30; or streptomycin, 10. Growth under methane was assessed after 1–2 weeks.
Enzyme assays.
To determine soluble methane monooxygenase (sMMO) activity, naphthalene oxidation by 1-week-old colonies grown on solid M medium without added copper was tested (Graham et al., 1992). Particulate methane monooxygenase (pMMO) activity was measured as 14CH4 consumption by whole cells (Sokolov & Trotsenko, 1995). For the preparation of cell-free extracts, exponentially grown cells were harvested by centrifugation at 6000 g for 30 min, and washed once in 50 mM Tris/HCl buffer (pH 7·5) containing 5 mM MgCl2. The cells were resuspended in the same buffer. Cells (0·5 g in 2 ml buffer) were sonicated three times for 30 s using an MSE sonifer (150 W, 20 kHz), in ice-cooled cuvettes. Cell debris was precipitated by centrifugation at 15 000 g for 30 min. The supernatant was used for enzyme assays, which were performed as described previously (Kalyuzhnaya et al., 1999).
Cellular fatty acid profile and phospholipid analyses.
For phospholipid analysis, exponentially grown cells at optimum temperature (59 °C) were harvested by centrifugation at 7000 g for 10 min. Phospholipids were determined according to the method described previously (Govorukhina & Trotsenko, 1989). Fatty acids and ubiquinones were analysed as described previously (Doronina et al., 2003).
DNA isolation and DNA G+C content determination.
DNA was isolated as described previously (Kalyuzhnaya et al., 1999). DNA G+C content was determined by using the thermal denaturation method (Sambrook et al., 1989).
Comparative sequence analysis.
For PCR of the 16S rRNA gene, the universal bacterial primers 27f and 1492r (Giovannoni, 1991) were used. Amplification of a partial fragment of the pmoA gene (encoding the 27 kDa peptide of pMMO) was done using primers A189 and mb661r (Bourne et al., 2001). Amplification of partial fragments of the mmoX gene (encoding the 
-subunit of sMMO) was performed using primers according to PCR programmes described by Murrell et al. (1998). Amplification of partial fragments of the nifH (encoding the iron protein of the nitrogenase) and cbbL (encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) genes was performed as described by Dedysh et al. (2004) and Baxter et al. (2002), respectively. PCR was performed using a Hybaid Thermocycler, with a mixture containing TaqPol buffer (USB), 0·2 µg chromosomal DNA, 0·5 µM oligonucleotide primers, 0·5 mM each dNTP, 2·5 mM MgCl2 and 2 U Taq polymerase. After denaturation at 95 °C for 2 min, the reaction mixture was subjected to 25 thermal cycles: 94 °C, 50 s; 50 s at annealing temperature (56 °C for pmoA gene-specific and 60 °C for 16S rRNA gene-specific primers); 72 °C, 50 s. At the final stage, the reaction mixture was incubated for 5 min at 72 °C. The size of the reaction products was checked on 1 % (w/v) agarose gels. For sequencing, the reaction products were separated by electrophoresis in 1 % low-melting point agarose (Gibco), and then the bands of corresponding size were excised and purified by using the agarase (Fermentas) digestion procedure, according to the manufacturer's protocol. The PCR products were sequenced by using a FemtoMol kit (Promega). Inferred polypeptide sequences of PmoA and 16S rRNA gene sequences were aligned manually with sequences retrieved from the GenBank database, using the CLUSTAL package of multiple alignment programs (Higgins & Sharp, 1988). Dendrograms were constructed using the TREECON programs package (version 1.3b) (Van de Peer & De Wachter, 1994).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Cultural and morphological characteristics
Small (1–2 mm in diameter), white, semi-transparent colonies with an entire edge and smooth surface were observed on agar plates after incubation at 53 °C for 1–2 weeks. Older colonies (1 month) became light brown and more rigid. The colonies grew faster and were relatively large (3 mm in diameter) when vitamins were added to the growth medium. As revealed by light microscopy, the cells grown in liquid culture in either exponential or stationary phase were mostly represented by coccoids of 0·6–0·8 µm in diameter (Fig. 1a). In contrast, the majority of cells grown on solid medium appeared as short rods, slightly varying in size (0·6–0·8x1·0–1·2 µm). The cells were non-motile, and multiplied by normal cell division. Electron microscopy analysis of ultrathin sections of cells showed a typical Gram-negative structure of the cell wall, and the presence of type I intracytoplasmic membrane (ICM) arranged as stacks of vesicular disks (Fig. 1b) that completely filled the cytoplasmic space. Glycogen inclusions and poly-
-hydroxybutyrate granules usually present in mesophilic methanotrophs were not observed. Also, the cells formed regularly arranged surface layers with p2 symmetry (Fig. 1c).
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Maximal growth rate occurred at 57–59 °C. Strain MYHTT was able to grow at 37 and 67 °C. Interestingly, the cells oxidized and assimilated methane even at 75 °C (see Supplementary Figure in IJSEM Online). The strain grew best at 57 °C in the presence of 0·5–1 % NaCl (specific growth rate 0·1 h–1 and generation time 7 h). The optimum NaCl concentration for growth at 65 °C was 0·5 % (w/v). The isolate was capable of growth in up to 3 % NaCl at 57 °C, but only in up to 1 % NaCl at 65 °C. The pH range for growth was 6·5–7·5. Cells oxidized methane most actively in the presence of 0·9–1·4 µM CuSO4. Higher copper concentrations (up to 2·4 µM) did not influence methane consumption.
Phospholipid fatty acid (PLFA) profiles
As can be seen from Table 1, strain MYHTT contained specific fatty acids, consisting mainly of C16 : 0 (37·24 %) and C18 : 19c (35·16 %). The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8.
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), suggesting that strain MYHTT assimilates methane carbon at the formaldehyde level via the ribulose monophosphate (RuMP) cycle. Neither hydroxypyruvate reductase (NADH) nor serine–glyoxylate aminotransferase activities were found, thus excluding the operation of the serine pathway. Also, no activity of the key enzyme of the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase, was found. The appropriate cbbL gene was not revealed by PCR. The complete tricarboxylic acid cycle does not operate in this organism because of the absence of -ketoglutarate dehydrogenase. Like many other methanotrophs, in strain MYHTT the pyrophosphate-dependent, but not the ATP-dependent, 6-phosphofructokinase was operative. Also, two possibilities for the cleavage of hexosephosphates exist, via fructose-1,6-bisphosphate aldolase and 2-keto-3-deoxy-6-phosphogluconate aldolase. The presence of glutamine synthetase and glutamate synthase activities indicated the involvement of the glutamate cycle in ammonia assimilation by strain MYHTT.
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). In contrast, the partial pmoA gene sequence of strain MYHTT exhibited no more than 77 % identity to the corresponding gene fragments (460 bp) of Methylocaldum szegediense OR2T, Methylocaldum gracile VKM-14LT and Methylococcus capsulatus Bath.
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). We have improved the growth of our isolate by optimization of the medium composition, by studying 14C-methane consumption and oxidation by cells. In contrast to ‘Methylothermus’ strain HB, isolate MYHTT was able to grow up to an OD600 value of 1·0, with a rather high growth rate (µ=0·1 h–1). Although the addition of vitamins stimulated the growth of both methanotrophs, these factors were excluded from the medium used for cultivation of strain MYHTT, to prevent its possible contamination and subsequent loss of the culture.
In principle, the colony and cell morphologies of strain MYHTT were similar to those of ‘Methylothermus’ strain HB (Bodrossy et al., 1999). Also, both organisms required the same level of CuSO4 for growth and lacked sMMO. However, the upper temperature limit for growth (72 °C) and optimum values (62–65 °C) were somewhat higher for ‘Methylothermus’ HB. Furthermore, the 16S rRNA gene similarity between strain MYHTT and ‘Methylothermus’ HB was low, indicating the affiliation of these strains at least to different species.
The metabolic pattern of strain MYHTT was quite different from those of the extant thermophilic/tolerant type X methanotrophs belonging to the genera Methylococcus and Methylocaldum, but was similar to that of the extremely halophilic Methylohalobius crimeensis (Table 3). Obviously, strain MYHTT uses only the RuMP cycle for carbon assimilation, whereas type X methanotrophs assimilate formaldehyde via both the RuMP and serine pathways, and are also able to grow autotrophically. These important differentiating properties allowed us to consider strain MYHTT to be a type I methanotroph (Table 3).
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9c in equal proportions, clearly distinguishes strain MYHTT from the other extant methanotrophs. Usually, C16 fatty acids prevail in PLFA profiles of type I and type X methanotrophs, whereas C18 fatty acids occur in very small amounts. In contrast, C18 fatty acids are predominant in type II methanotrophs. Only the newly isolated halophilic methanotroph Methylohalobius crimeensis belonging to the type I methanotrophs had a large proportion (51·87 %) of C18 : 17c (Heyer et al., 2005). Analysis of the 16S rRNA and pmoA genes clearly showed that strain MYHTT, together with ‘Methylothermus’ strain HB and Methylohalobius crimeensis, represent a deep-branching group of extremophilic methanotrophs, which might indicate their common origin and consequent divergence. Therefore, strain MYHTT appears to be more closely related to the type I (mesophilic, halophilic methanotroph Methylohalobius) than to the type X (thermophilic/tolerant Methylococcus and Methylocaldum) methanotrophs. Because the published data on the properties of ‘Methylothermus’ strain HB are not sufficient for a comprehensive comparison with those of strain MYHTT, and also because strain HB is not available, we propose that strain MYHTT represents a new genus, Methylothermus gen. nov. Based on the phenotypic and genotypic properties, and phylogeny, we propose that isolate MYHTT should be classified as representing the type species of the genus Methylothermus, with the name Methylothermus thermalis sp. nov.
Description of Methylothermus gen. nov.
Methylothermus (Me.thy'lo.ther'mus. N.L. n. methyl the methyl group; N.L. masc. subst. from Gr. adj. thermos hot; Methylothermus methyl-using thermotolerant organism).
Cells are Gram-negative, non-motile coccoids, of 0·6–0·8 µm in diameter. Reproduce by binary division. Exospores, Azotobacter-type cysts or lipid cysts are not formed. Possess pMMO and type I ICM as stacks of membrane vesicles. Do not possess sMMO. Moderate thermophiles growing at 37–67 °C (optimum 57–59 °C). Obligate methanotrophs utilizing methane or methanol via the RuMP pathway. Tricarboxylic acid cycle is deficient in -ketoglutarate dehydrogenase. Major PLFAs are C16 : 0 and C18 : 19c. Major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Phylogenetically belong to the class ‘Gammaproteobacteria’ (type I methanotrophs). Habitats are hot springs. The type species is Methylothermus thermalis.
Description of Methylothermus thermalis sp. nov.
Methylothermus thermalis (ther'mal.is. N.L. masc. adj. thermalis pertaining to a hot spring).
Description is as for the genus with the following amendments. Grows on nitrate, ammonia, urea, tryptophan, lysine, glutamine, formamide and Tris as a nitrogen source. Is not able to fix atmospheric nitrogen. Prefers media containing 0·5–1 % NaCl and is capable of growth in 3 % NaCl. Neutrophile, growing at pH 6·5–7·5. DNA G+C content is 62·5 mol%. The type strain is MYHTT (=VKM B-2345T=IPOD FERM P-19714T), which was isolated from a hot spring located in Hyogo, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Bodrossy, L., Murrell, J. C., Dalton, H., Kalman, M., Puskas, L. G. & Kovacs, K. L. (1995). Heat-tolerant methanotrophic bacteria from the hot-water effluent of a natural-gas field. Appl Environ Microbiol 61, 3549–3555.[Abstract]
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Bowman, J. P., Sly, L. I., Nichols, P. D. & Hayward, A. C. (1993). Revised taxonomy of the methanotrophs: description of Methylobacter gen. nov., emendation of Methylococcus, validation of Methylosinus and Methylocystis species, and a proposal that the family Methylococcaceae includes only the group I methanotrophs. Int J Syst Bacteriol 43, 735–753.
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