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1 S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia
2 Netherlands Institute of Ecology, NL3631 AC Nieuwersluis, The Netherlands
3 G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia
4 Joint Graduate School of Energy and Environment, King Mongkut's University of Technology, Bangkok 10140, Thailand
5 Max-Planck-Institut für terrestrische Mikrobiologie, D-35043 Marburg, Germany
6 Institute of Geological and Nuclear Sciences, Wairakei Research Centre, Taupo, New Zealand
Correspondence
Svetlana N. Dedysh
dedysh{at}mail.ru
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8c), the cells also contained large amounts of what was previously considered to be a signature fatty acid of type I methanotrophs, 16 : 18c. The DNA G+C contents of strains H2T and Sakb1 were 61.5 and 62.1 mol%, respectively. The 16S rRNA gene sequences possessed 96–98 % similarity to sequences of other type II methanotrophs in the genera Methylosinus and Methylocystis. 16S rRNA gene sequence and pmoA phylogeny demonstrated that the strains form a novel lineage within the genus Methylocystis. DNA–DNA hybridization values of strain H2T with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T were 18 and 25 %, respectively. Thus, it is proposed that these two strains represent a novel species, Methylocystis heyeri sp. nov. Strain H2T (=DSM 16984T=VKM B-2426T) is the type strain.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and partial sequences of the mxaF, mmoX and pmoA genes of Methylocystis heyeri strain H2T are AM283543–AM283546, respectively.
A supplementary figure showing mass spectra of DMDS adducts of 16 : 1 PLFA of strain H2T is available in IJSEM Online.
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). Only two of the 13 currently recognized genera of methanotrophs, Methylocella and Methylocapsa, are acidophilic (Dedysh et al., 2000, 2002). However, molecular identification of methanotroph populations in acidic Sphagnum-dominated peatlands (McDonald & Murrell, 1997; Dedysh et al., 2001, 2003) and acidic forest soils (Radajewski et al., 2002; Knief et al., 2005) has suggested that uncultivated subgroups within the genus Methylocystis are also capable of growth under acidic conditions. In this paper, two novel Methylocystis strains isolated from two acidic environments, Sphagnum peat and forest soil, are described. A novel species is proposed for these acidophilic methanotrophs.
Strain H2T was isolated from a sample collected in July 2001 from 10 cm below the surface of Sphagnum peat (pH 4.3) on the bank of the bog lake Teufelssee in north-eastern Germany. A methanotrophic enrichment culture was obtained using liquid medium M2 at pH 5.5 as described by Dedysh et al. (1998). To identify methanotrophs in this enrichment, a whole-cell hybridization was applied with a set of 16S rRNA-targeted oligonucleotide probes developed for differential detection of type II methanotrophs (Dedysh et al., 2003). Most of the cells in the enrichment hybridized to probe Mcyst-1261, which was designed to target a peat-inhabiting subgroup of Methylocystis species. Attempts to isolate these methanotrophs in pure culture by plating on agar medium M2 were unsuccessful. However, replacement of agar with gellan gum (Gel-Gro; ICN Biomedicals) resulted in the growth of methanotroph colonies and allowed us to obtain strain H2T in a pure culture. Another strain, Sakb1, was isolated using diluted nitrate mineral salts (DNMS) medium of pH 5.8 from an acidic (pH 4.2) evergreen forest soil in Thailand (Knief et al., 2005).
The isolates were maintained on their respective agar media (medium M2 for strain H2T and DNMS medium for strain Sakb1) and in liquid cultures. For growth in liquid media, 500 ml screw-cap serum bottles were used with a headspace/liquid space ratio of 4 : 1. After inoculation, the bottles were sealed with silicon rubber septa and methane was added aseptically using a syringe equipped with a disposable filter (0.22 µm) to achieve a 10–20 % mixing ratio in the headspace. Bottles were incubated on a rotary shaker (120 r.p.m.) at 24 °C. The absence of heterotrophic satellites was checked by phase-contrast microscopy and by plating methanotrophic isolates on 10-fold diluted Luria–Bertani agar (1.0 % tryptone, 0.5 % yeast extract, 1.0 % NaCl) or M2 agar medium amended with 0.1 % (w/v) glucose. Morphological observations, examination of thin sections, tests for utilization of different carbon and nitrogen sources and enzyme assays were performed as described previously (Dedysh et al., 2000; Dunfield et al., 2003). Growth of isolates in liquid culture with methane as the sole growth substrate was monitored by nephelometry at 410 nm for 2 weeks under a variety of growth conditions, including temperatures of 4–37 °C, pH values of 4.1–8.0 and NaCl concentrations of 0.01–2.00 % (w/v).
For fatty acid analyses, cells of strains H2T and Sakb1 were grown on their respective liquid mineral media under methane and harvested in the late exponential phase. Lipids were extracted from 2.6 mg (strain Sakb1) or 6 mg (strain H2T) freeze-dried cell material using a modified Bligh and Dyer extraction procedure (Boschker et al., 1998, 2001). The lipid extract was fractionated on silicic acid into different polarity classes by sequential elution with chloroform, acetone and methanol. The methanol fraction containing the PLFA was derivatized using mild-alkaline methanolysis to yield fatty acid methyl esters (FAMEs). FAME standards of C12 : 0 and C19 : 0 were used for calculating retention indices and for quantification. Identification of FAMEs was based on retention time data against known standards. For identification of methanotroph-specific PLFA, type culture extracts (Methylomonas methanica NCIMBT 11130; Methylomicrobium album NCIMB 11123T; Methylobacter luteus NCIMB 11914T; Methylocystis parvus NCIMB 11129T; Methylosinus trichosporium NCIMB 11131T; Methylosinus sporium NCIMB 11126T) were used as references. FAME concentrations were determined using a GC-FID system (Thermo Finnigan TRACE GC) equipped with a polar capillary column (SGE, BPX-70, 50 mx0.32 mmx0.25 µm) using the following oven conditions: initial temperature 50 °C for 1 min, then increasing to 130 °C by 40 °C min–1, then increasing to 230 °C by 3 °C min–1.
To determine double bond position of monounsaturated PLFAs, dimethyldisulfide (DMDS) derivatization was performed as described by Nichols et al. (1986). DMDS adducts were analysed on a Finnigan TRACE GC-MS system. DMDS adducts were separated using an Rtx-5MS (60 mx0.32 mmx0.25 µm) capillary column with the following oven conditions: initial temperature 80 °C for 1 min, then increasing to 160 °C by 40 °C min–1, then increasing to 330 °C by 4 °C min–1. MS operating parameters were: electron multiplier 375 V; source temperature 200 °C; transfer line temperature 250 °C; fullscan m/z 33–410. MS data were acquired and processed using the Finnigan Xcalibur software.
DNA was extracted from strains H2T and Sakb1 using an SDS-based procedure (Dedysh et al., 1998). The G+C content of the DNA was determined by thermal denaturation (Owen et al., 1969) using a Unicam SP1800 spectrophotometer at a heating rate of 0.5 °C min–1. The DNA of Escherichia coli K-12 was used as the standard. DNA–DNA hybridization of strain H2T with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T was performed as described by Lysenko et al. (1988). PCR-mediated amplifications of the nearly complete 16S rRNA gene and partial fragments of the following genes (expressed protein also given) were performed: mmoX [
-subunit of soluble methane monooxygenase (sMMO)]; mxaF [large subunit of methanol dehydrogenase (MDH)] and pmoA [
-subunit of particulate methane monooxygenase (pMMO)]. The products were sequenced as described by Heyer et al. (2002). Phylogenetic sequence analyses were carried out using the ARB program package (Ludwig et al., 2004).
Cells of isolates H2T and Sakb1 were Gram-negative and non-motile. Strain H2T formed straight polymorphic rods or ovoids (Fig. 1a), whereas strain Sakb1 formed regularly curved rods (Fig. 1b). Old cultures of strain H2T contained many misshapen and forked cells, whereas cells of strain Sakb1 attained a distinct crescent shape, almost to a closed circle. Cells were 0.8–1.2 µm wide and 1.4–4.0 µm long, did not form rosettes during any growth stage and reproduced by normal cell division. When grown on solid media, cells produced large capsules up to 3.0–4.0 µm thick, so that each cell was separated from other cells due to the extensive capsular material (Fig. 1a, b). No exospores or cysts were observed regardless of cell age or culture conditions. On DNMS agar, strain Sakb1 formed large (up to 1–2 cm in diameter), raised, white, slimy colonies with an entire edge and a smooth surface. Strain H2T did not develop on agar media but on solid media made with Gel-Gro, colonies were similar to those of strain Sakb1, although smaller (up to 0.5 cm in diameter). Liquid cultures of both strains displayed white turbidity. No surface pellicle or cellular aggregates were formed. Analysis of thin sections of cells grown on methane revealed a well-developed system of ICM aligned parallel to the cytoplasmic membrane (Fig. 1c). This ICM arrangement is typical of members of the Methylosinus/Methylocystis group of type II methanotrophs.
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The isolates grew at pH 4.4–7.5, with optimum growth at pH 5.8–6.2. This optimum and range are lower that those of other members of the genera Methylocystis and Methylosinus (Bowman et al., 1993). The temperature range for growth was 5–30 °C, with optimum growth at 25 °C. No growth occurred at 37 °C. The specific growth rate of strain H2T in liquid culture under CH4 (10 %, v/v) at optimal temperature and pH was 0.09 h–1 (equal to a doubling time of 7.7 h). Both strains grew best on diluted media. Growth inhibition of 50 % was observed in the presence of 0.5 % NaCl (w/v) and 0.8 % NaCl completely inhibited growth.
The PLFA profiles of strains H2T and Sakb1 deviated substantially from those of other type II methanotrophs (Table 1). The major component of these profiles was 18 : 18c fatty acid. This fatty acid is common and specific to the genera Methylosinus and Methylocystis (Guckert et al., 1991; Bowman et al., 1993), but it is lacking in cells of members of the genera Methylocella and Methylocapsa (Dedysh et al., 2002, 2004; Dunfield et al., 2003). However, several other PLFAs were detected that are atypical of Methylocystis/Methylosinus. One of these was C18 : 19t, which comprised 14 % of the total PLFA content in strain H2T. An even more surprising component was 16 : 18c, which comprised 25 and 29 % of the total PLFAs in Sakb1 and H2T, respectively. This PLFA has previously been detected only in type I methanotrophs (Gammaproteobacteria) and has been regarded as a specific signature lipid for this group (Hanson & Hanson, 1996). Besides 16 : 18c, the novel strains also contained 16 : 15t (2.75 % in H2T), another PLFA previously found to occur only in type I methanotrophs.
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8c in type II methanotrophs was unexpected, this result was verified via DMDS derivatization. Fig. 2 and Supplementary Fig. S1 (available in IJSEM Online) show the MS chromatograms. The total ion chromatogram (Fig. 2a) shows essentially the same output as the GC-FID chromatogram. The peak at retention time 32.84 (Fig. 2a) and 32.91 (Fig. S1a) was identified as 16 : 18c. In the DMDS procedure, a mass fragment m/z 203 (diagnostic for 16 : 18c) was formed. This comprises that part of the PLFA molecule that includes the carboxylic (
) end. In Fig. 2c, the m/z 203 -fragment matched the peak in the total ion chromatogram in Fig. 2a, which was identified as 16 : 18c by retention time. This identification was also confirmed using the -fragments of the DMDS adducts. The presence of a second ‘signature’ lipid of type I methanotrophs, 16 : 15t, was also detected by GC-FID and DMDS analyses in both strains (Fig. 2f). This lipid was not detectable in the total ion chromatogram because the abundance of the component, indicated by the intensity of the fragment, was low. The PLFA 16 : 17c could not be separated completely from 16 : 18c in the GC-MS run because the DMDS adducts were analysed on an apolar column. Using the polar column for GC-FID, all compounds were separated. However, a comparison of the intensities in Fig. 2a and Fig. 2d shows that the peaks at 32.91 and 32.84, respectively, comprise the majority of the C16 : 18c peak.
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8c fatty acid makes these strains unique among described type II methanotrophs. Interestingly, significant amounts of 16 : 18c fatty acid were detected in extracts made from acidic peat (Krumholz et al., 1995; Sundh et al., 1995). This was interpreted to mean that type I methanotrophs were abundant. In contrast, in situ hybridization with methanotroph-specific oligonucleotide probes detected only relatively small populations of type I methanotrophs in acidic peat (Dedysh et al., 2001, 2003). The data presented in this study provide a possible explanation for this controversy and suggest that there are severe difficulties with the use of 16 : 18c as a specific biomarker for type I methanotrophs.
The metabolic patterns of strains H2T and Sakb1 were similar to those of other members of the genera Methylosinus and Methylocystis (Table 2). As demonstrated by the production of naphthol from naphthalene, both strains possessed sMMO. High activities of hydroxypyruvate reductase and serine-glyoxylate aminotransferase indicated that strains H2T and Sakb1 assimilate C1 compounds via the serine pathway. Activities of Calvin cycle enzymes (phosphoribulokinase and ribulose-bisphosphate carboxylase) or the key ribulose monophosphate cycle enzyme (hexulose phosphate synthase) were not found. The presence of 2-oxoglutarate dehydrogenase indicates that the complete tricarboxylic acid cycle operates in this bacterium. Glutamate cycle enzymes, glutamine synthetase and glutamate synthase, were used for ammonium assimilation.
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). The environmental clone sequences UP4, UP5, UP6 and UP7 that were retrieved from an acidic forest soil (Radajewski et al., 2002) shared an overall 16S rRNA gene sequence similarity of 98 % with the novel isolates. Differences between the novel isolates and the type strains of related species were: Methylocystis parvus OBBPT (2.6–3.1 %), Methylocystis echinoides IMET 10491T (2.6–3.1 %), Methylocystis rosea SV97T (2.4–3.1 %), Methylosinus trichosporium NCIMB 11131T (3.5–3.6 %) and Methylosinus sporium NCIMB 11126T (3.4–3.5 %). Isolates H2T and Sakb1 were generally more similar to species of the genus Methylocystis than to those of the genus Methylosinus. However, if all available sequences of different strains described by Heyer et al. (2002) are included in treeing analyses, 16S rRNA gene sequence phylogeny is variable depending on the algorithms used and the two genera are not often distinguished. Phylogenetic analysis based on partial pmoA sequences is better for revealing the species and genus relationships of type II methanotrophs (Heyer et al., 2002). The pmoA phylogeny clearly showed that the novel strains represent a lineage that is distinct from all representatives of both genera, but closer to Methylocystis (Fig. 4).
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8c in PLFA profiles and results of phylogenetic analyses place strains H2T and Sakb1 within the type II methanotroph group. Cell morphology, the absence of exospores and rosettes and the phylogeny of pmoA identify the novel strains as members of the genus Methylocystis. However, compared with recognized representatives of the genus Methylocystis, the novel strains differ in cell shape and by the formation of large exopolysaccharide capsules, in their preference for moderately acidic conditions and in their unique PLFA profile. The DNA–DNA hybridization values of strain H2T with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T were 18 and 25 %, respectively. Thus, it is concluded that strains H2T and Sakb1 represent a novel species of the genus Methylocystis, for which the name Methylocystis heyeri sp. nov. is proposed.
Emended description of the genus Methylocystis (ex Whittenbury et al. 1970) Bowman et al. 1993
Methylocystis [Me.thy.lo.cys'tis. N.Gr. n. methyl (from Gr. n. methu wine and Gr. n. hulê wood) methyl group; N.L. fem. n. cystis (from Gr. fem. n. kustis) the bladder and in biology, a cyst; N.L. fem. n. Methylocystis methyl cyst].
Gram-negative cells that are reniform, coccobacillary or rod-shaped; 0.3–1.2 µm wide by 0.5–4.0 µm long. Reproduces by normal cell division. Does not form either rosettes or exospores. May form a lipid cyst. Non-motile. Encapsulated. May accumulate poly--hydroxybutyrate and polyphosphate. May form spinae on cell surfaces. Contains type II ICMs that are aligned parallel to the cell wall. Possesses pMMO; some strains may possess sMMO. Grows at temperatures between 5 and 40 °C and at pH values between 4.5 and 9.0. Organic growth factors and NaCl are not required for growth. Aerobic chemolithotroph. Obligate utilizer of C1 compounds via the serine pathway. No growth occurs on complex organic media. Does not contain the Benson–Calvin cycle for CO2 fixation, but contains a complete tricarboxylic acid cycle. Capable of dinitrogen fixation. Produces oxidase and catalase. All representatives possess 18 : 18c as the predominant PLFA; some species may also possess 16 : 18c as a major PLFA. The major quinone is ubiquinone 8. The DNA G+C content ranges from 61.5 to 67.0 mol%. Phylogenetically belongs to the Alphaproteobacteria and is closely related to the genus Methylosinus. Methylocystis parvus is the type species.
Description of Methylocystis heyeri sp. nov.
Methylocystis heyeri (hey.e'ri. N.L. gen. n. heyeri of Heyer, named in honour of the German microbiologist Jürgen Heyer, for his highly enthusiastic research on methanotrophic bacteria and his contributions to increasing our knowledge on methanotroph diversity and ecology).
Cells are large, straight or regularly curved rods or ovoids; 0.8–1.2 µm wide and 1.4–4.0 µm long. Old cultures may contain misshapen and forked cells or nearly closed crescents. Cells possess large capsules so that each cell is separated from the others by plenty of the capsular material. Cysts are not formed. Colonies are white, highly raised and slimy. Liquid cultures display homogeneous turbidity. Grows at temperatures between 5 and 30 °C (optimum at 25 °C) and at pH values between 4.4 and 7.5 (optimum at 5.8–6.2). Carbon sources include methane and methanol. Methanol is utilized at concentrations below 1 % (v/v); optimal growth occurs at 0.1 % (v/v) methanol. NaCl inhibits growth at concentrations above 0.5 %. The predominant PLFAs are 18 : 18c and 16 : 18c.
The type strain, H2T (=DSM 16984T=VKM B-2426T), was isolated from the acidic Sphagnum peat bog lake Teufelssee, north-eastern Germany. Strain Sakb1 is a reference strain, isolated from acidic tropical forest soil (Thailand). The DNA G+C contents of strains H2T and Sakb1 are 61.5 and 62.1 mol%, respectively.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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