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1 Department of Biology, Faculty of Science, University of Tromsø, N-9037 Tromsø, Norway
2 Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand
Correspondence
Mette M. Svenning
Mette.Svenning{at}ib.uit.no
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8, 16 : 17 and 16 : 15t) supported the affiliation of strain SV96T to the genus Methylobacter. The results of DNA–DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV96T from the four Methylobacter species mentioned above. Strain SV96T therefore represents a novel species, for which the name Methylobacter tundripaludum sp. nov. is proposed (type strain SV96T=DSM 17260T=ATCC BAA-1195T).
Published online ahead of print on 26 August 2005 as DOI 10.1099/ijs.0.63728-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, pmoA and nifH sequences of Methylobacter tundripaludum SV96T are AJ414655, AJ414658 and AY937260, respectively.
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), and Methylobacter species are equivalent to the similarly named group first coined by Whittenbury et al. (1970). The genus was further revised when three members of the genus Methylobacter were renamed as a new taxon Methylomicrobium (Bowman et al., 1995). At present the genus comprises the four species Methylobacter luteus (Bowman et al., 1993; Romanovskaya et al., 1978), Methylobacter whittenburyi (Bowman et al., 1993; Romanovskaya et al., 1978; Whittenbury et al., 1970), Methylobacter marinus (Bowman et al., 1993; Lidstrom, 1988) and Methylobacter psychrophilus (Omelchenko et al., 1996; Tourova et al., 1999).
Strain SV96T was isolated from a soil core collected from a wetland near the settlement Ny-Ålesund (78° 56' N 11° 53' E), Svalbard, in July 1996. The soil emitted methane (Høj et al., 2005) and had a pH of 6·4. The temperature of the soil was 10 °C at the surface and 5 °C at 10 cm below the surface; the permafrost level was at 25 cm. After the fresh vegetation layer was removed, the upper 10 cm of the soil core was mixed, 2 g soil was taken and added to 10 ml nitrate mineral salt medium (NMS) (Whittenbury et al., 1970) at pH 6·8, and shaken for 10 min at 200 r.p.m. After 10 min of sedimentation, 1 ml supernatant was mixed with 9 ml NMS in a 120 ml serum bottle (Alltech). The bottle was sealed with a rubber stopper and crimp cap. Twenty millilitres of air was replaced with 20 ml CH4/CO2 (95 : 5) mixture (both gases were 99·95 % purity). The bottle was incubated at 20 °C and subcultured every 2–3 weeks (1 ml culture to 9 ml fresh NMS medium). After four or five subculturing steps in liquid media, bacteria were plated on NMS medium containing 1·5 % noble agar (bacteriological agar type E; Biokar diagnostics). The plates were incubated at 20 °C in sealed chambers containing approximately 35 % methane in air. Colonies were picked and restreaked. Heterotrophic contamination was checked by streaking colonies on agar plates with rich medium containing 0·5 % tryptone, 0·25 % yeast extract, 0·1 % glucose and 2·0 % agar. These plates were incubated at room temperature without additional methane. The cultures were considered to be pure when only one cell type was observed under light microscopy and no growth on nutrient rich medium was observed. Exospore formation was assayed by determining cell viability after heating 3-week-old cultures to 80 °C for 20 min and then observing microcolony formation on NMS agar after 2 weeks incubation with methane (Bowman et al., 1993). Cyst formation was assayed by the method of Vela & Wyss (1964). Tolerance to NaCl concentrations ranging from 0·01 to 1·0 % (w/v) was determined with NMS agar cultures. Growth at pH values ranging from 5·5 to 9·0 (pH was adjusted with NaOH or HCl) and methanol concentrations ranging from 0·1 to 1·0 % (v/v) was determined in liquid cultures of NMS.
The growth of SV96T was measured at temperatures ranging from 5·0 to 40·0 °C, using a temperature gradient apparatus with the opportunity to grow bacteria simultaneously at 10 different chosen temperatures. The temperature gradient was achieved using a metal cylinder with a flat top (14 cm in diameter and 50 cm in length), where one end was attached to a cooling water bath and the other to a thermostat-regulated heat block. The top of the cylinder had 10 holes, 5 cm apart in two parallel rows, which fitted a 27 ml serum bottle (Alltech). The temperatures were measured at every second hole, where a small hole was drilled between the two parallel rows. To achieve temperatures from 5 to 40 °C, two different experiments with partly overlapping temperatures were run. The temperature was first set from 5 to 12 °C (a gradient of 0·14 °C cm–1) and then from 11 to 40 °C (a gradient of 0·58 °C cm–1). Cultures of SV96T were prepared by adding 5 ml starting culture to 27 ml serum bottles. The bottles were sealed with rubber stoppers and crimp caps before 5 ml air was replaced with 5 ml CH4/CO2 (95 : 5) mixture and the cultures were grown for 5 days. The OD600 was measured for the starting and 5-day-old cultures using a Spectramax 250 microplate spectrophotometer system (Molecular Devices). The maximum OD600 of SV96T was 0·72, and was measured after 15 days at 20 °C. The experiment was repeated in three replicates at each temperature setting and the net growth calculated by subtracting the OD600 of the starting culture from the OD600 values of the 5-day-old cultures. Mean values for net growth and standard deviations were calculated.
The 16S rRNA gene was analysed as described previously (Lane, 1991). Phylogenetic analysis was performed using the software packages PHYLIP (Felsenstein, 1993) and TREEVIEW (Page, 1996) after multiple alignment of data by CLUSTAL_X (Thompson et al., 1997). Distances (Kimura two-parameter model) and clustering by neighbour-joining methods were determined by using bootstrap values based on 100 replicates (Fig. 1). The 16S rRNA gene sequence of strain SV96T was a continuous stretch of 1487 bp. Sequence similarity calculations indicated that the closest relatives of strain SV96T were Methylobacter psychrophilus Z-0021T (99·1 %), Methylobacter luteus ATCC 49878T (97·3 %), Methylobacter marinus A45T (97·0 %) and Methylobacter whittenburyi ATCC 51738T (95·8 %). Lower sequence similarities were found with all species with validly published names of the genus Methylomicrobium: Methylomicrobium album ATCC 33003T (95·1 %), Methylomicrobium agile ATCC 35068T (94·5 %) and Methylomicrobium pelagicum NCIMB 2265T (94·0 %). The pmoA gene was analysed as described previously (McDonald & Murrell, 1997). Phylogenetic analysis and alignment of data were performed using the software package ARB (Ludwig et al., 2004) (Fig. 2). The nifH gene was amplified and sequenced as described previously (Poly et al., 2001). Sequence similarity searches in GenBank indicated that the closest related nifH sequence was derived from an uncultured N2-fixing bacteria (AY196439) (92 %); the closest matches with cultured bacteria were Methylobacter marinus A45T (84 %) and Methylobacter luteus NCIMB 11914T (=ATCC 49878T) (84 %). The DNA base composition was determined by thermal denaturation using a Cary 4E Varian spectrophotometer at a heating rate of 0·5 °C min–1. The G+C content (mol%) of the DNA was calculated by the equation of Mandel et al. (1970). DNA–DNA hybridization was performed with Methylobacter luteus ATCC 49878T according to the method described by Ezaki et al. (1989). It was not performed with Methylobacter psychrophilus Z-0021T or Methylobacter marinus A45T because they were, to our knowledge, both unavailable from any culture collection.
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), sharing 99·1 % 16S rRNA gene similarity, was isolated from a moss-vegetated area on the tundra in the polar Ural (Omelchenko et al., 1993). Despite the high 16S rRNA gene sequence similarity and the similar origin, the bacterial strains were different in several important morphological traits (Table 1). SV96T was non-motile, 0·8–1·3 µm wide and 1·9–2·5 µm in length. Cells often appeared in pairs or in long chains. Colonies were pale-pink pigmented, but lost the pigmentation upon methane starvation. Cells of Methylobacter psychrophilus Z-0021T were reported to be cocci or diplococci, 1·0–1·7 µm in diameter, and colonies were opaque cream (Omelchenko et al., 1996). The cells of SV96T lysed in 2 % SDS. The strain did not grow when NaCl was added to solid NMS medium and poor to no growth was observed with methanol as a carbon source. SV96T grew at all pH values tested except for pH 9·0 and no exospores or cysts were revealed using the described methods. A type I membrane structure, with bundles of disc-shaped membranes, was confirmed using transmission electron microscopy (Fig. 3). The major phospholipid fatty acids (PLFAs) for SV96T were 16 : 18 (34·9 %), 16 : 17 (23·4 %) and 16 : 15t (26·3 %). Isolate SV96T had a G+C content of 47 mol% (±1 mol%), while Methylobacter psychrophilus Z-0021T had a G+C content of 45·6 mol% (Omelchenko et al., 1996). SV96T was positive for the nifH gene by PCR (Poly et al., 2001). It was negative for the mmoX gene in PCR assays (Fuse et al., 1998; Miguez et al., 1997) and no soluble methane monooxygenase (sMMO) was detected by colorimetric assay performed as described by Brusseau et al. (1990), with the modifications of Graham et al. (1992). SV96T had an optimum growth temperature of 23 °C, but grew well at temperatures from 10 to 27 °C (Fig. 4) and was clearly not psychrophilic. Methylobacter psychrophilus Z-0021T was reported to have a growth optimum between 3·5 and 10 °C with no growth at temperatures above 20 °C (Omelchenko et al., 1996). To confirm that SV96T and Methylobacter psychrophilus Z-0021T represent different species, a DNA–DNA hybridization should be performed; however, Methylobacter psychrophilus Z-0021T is not available from any culture collection (P. Kämpfer, personal communication). This is also the situation for Methylobacter marinus A45T. The 16S rRNA gene sequence similarity between SV96T and the second most closely related bacterium, Methylobacter luteus ATCC 49878T, was 97 %. DNA–DNA hybridization demonstrated a relatedness value of 10 % between SV96T and Methylobacter luteus ATCC 49878T. The differences between Methylobacter luteus ATCC 49878T, Methylobacter psychrophilus Z-0021T, Methylobacter marinus A45T, Methylobacter whittenburyi ATCC 51738T and SV96T are described in Table 1. Because of the genotypic and phenotypic differences between SV96T and the other Methylobacter species (including 16S rRNA gene sequence and DNA–DNA hybridization), we propose the name Methylobacter tundripaludum sp. nov. for strain SV96T.
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Gram-negative, straight, fat, rod-shaped cells. Cells are 0·8–1·3 µm in diameter and 1·9–2·5 µm long. Cells are pale-pink pigmented, occur singly, in pairs or in long chains, and are non-motile. Grows on methane as sole carbon and energy source; shows poor to no growth on methanol. Possesses a type I intracytoplasmic membrane system. Assimilates carbon via the ribulose monophosphate (RuMP) pathway and does not possess sMMO. Cells are catalase- and oxidase-positive. Possesses a nifH gene. Optimal growth occurs at 23 °C, but grows well down to 10 °C; no growth occurs above 30 °C. Does not require NaCl for growth and cells are lysed by 2 % SDS. Grows well from pH 5·5 to 7·9. Major PLFAs are 16 : 18 (34·9 %), 16 : 17 (23·4 %) and 16 : 15t (26·3 %). The DNA G+C content is 47 mol%. Methylobacter tundripaludum strain SV96T shows 10 % DNA–DNA hybridization with Methylobacter luteus ATCC 49878T.
The type strain is SV96T (=ATCC BAA-1195T=DSM 17260T), isolated from an Arctic wetland soil near Ny-Ålesund, Svalbard.
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ACKNOWLEDGEMENTS |
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