| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
8 as the major fatty acid, non-motile, no rosette formation) supported the affiliation of strain SV97T to the genus Methylocystis. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain SV97T from the two recognized Methylocystis species. Strain SV97T therefore represents a novel species, for which the name Methylocystis rosea sp. nov. is proposed, with the type strain SV97T (=DSM 17261T=ATCC BAA-1196T).
-hydroxybutyratePublished online ahead of print on 28 October 2005 as DOI 10.1099/ijs.0.63912-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, pmoA and nifH gene sequences of Methylocystis rosea SV97T are AJ414656, AJ414657 and DQ010107, respectively.
A chart showing the growth characteristics of Methylocystis rosea SV97T at different temperatures and a detailed description of the methods used for fatty acid analysis are available as supplementary material in IJSEM Online.
Present address: Svanhovd Miljøsenter, N-9925 Svanvik, Norway. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
), Methylocella (Dedysh et al., 2000) and Methylocapsa (Dedysh et al., 2002). They are distinguished from type I methanotrophic bacteria by genetic and phenotypic features. Type II methanotrophs belong to the Alphaproteobacteria, assimilate formaldehyde by the serine pathway, have intracytoplasmic membranes aligned parallel to the cell wall (Methylocystis and Methylosinus), membrane vesicles packed in parallel on one side of the cell membrane (Methylocapsa) or a vesicular membrane system connected to the cytoplasmic membrane (Methylocella), and the major fatty acid is C18 : 18c (Methylocystis and Methylosinus) (Hanson & Hanson, 1996) or C18 : 17c (Methylocella and Methylocapsa) (Dedysh et al., 2000, 2002). At present, the genus Methylocystis comprises two species with validly published names, Methylocystis parvus (Bowman et al., 1993; Whittenbury et al., 1970) and Methylocystis echinoides (Galchenko et al., 1977). However, a large number of strains designated as members of the genus Methylocystis has been isolated from several different environments (Dunfield et al., 2002; Heyer et al., 2002; Wise et al., 1999).
Strain SV97T was isolated from a soil core collected from a high Arctic wetland near the Ny-Ålesund settlement (78° 56' N 11° 53' E) on the Svalbard Islands, Norway, in July 1997. At the time of sampling, the soil had a pH of 6·4 and the temperature of the soil was 10 °C at the surface and 5 °C at 10 cm below the surface and the permafrost level was at 25 cm. Methane emission from the same site was determined in July and September 1998 and in July and August 1999 and emission varied from 93 to 2801 µg CH4 m–2 h–1 (Høj et al., 2005).
After removal of the fresh vegetation layer, the upper 10 cm of the soil core was mixed and 2 g soil was added to 10 ml nitrate mineral salt (NMS) medium (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 medium in a 120 ml serum bottle (Alltech) and then sealed with a rubber stopper and crimp cap. The gas atmosphere inside the bottle was enriched with methane by replacing 20 ml air with 20 ml CH4/CO2 (95 : 5) mix. 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 20 °C 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 or on NMS agar without methane 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) and by light microscopy of 3-week-old cultures. The presence of poly--hydroxybutyrate (PHB) inclusions was determined by staining with 0·03 % (w/v) Sudan black and 0·5 % safranin (Gerhardt, 1981) and observing using phase-contrast microscopy. The presence of PHB was also confirmed by chemical analysis (Gerhardt, 1981). Tolerance of NaCl concentrations ranging from 0·01 to 1·0 % (w/v) was determined with NMS agar cultures. Growth at pH range 5·5–9·0 was determined in liquid cultures of NMS. The pH of the medium was adjusted with NaOH or HCl. Growth on alternative carbon sources was determined on methanol concentrations ranging from 0·1 to 1·0 % (v/v) and ethanol, formate, formamide, formaldehyde, glucose, sucrose, acetate, citrate and yeast extract, all at 0·1 % (v/v), in liquid cultures of NMS. Ability to fix nitrogen was determined by growth on nitrate (NMS), ammonia (AMS) and nitrogen-free (NFMS) media (Auman et al., 2001). Possession of the soluble methane monooxygenase (sMMO) was tested for by using a colorimetric assay described by Brusseau et al. (1990), with modifications (Graham et al., 1992). The DNA G+C content was determined by thermal denaturation using a spectrophotometer (Cary 4E; Varian) at a heating rate of 0·5 °C min–1. The DNA G+C content was calculated as described by Mandel et al. (1970). DNA–DNA hybridization was performed with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T according to Ezaki et al. (1989). The 16S rRNA gene, soluble methane monooxygenase gene (mmoX), small subunit of particulate methane monoxygenase gene (pmoA) and nitrogenase reductase structural gene (nifH) were analysed by PCR as described previously (Fuse et al., 1998; Miguez et al., 1997).
Growth of strain SV97T was measured at temperatures ranging from 5 to 40 °C, using a temperature gradient apparatus. Cultures of SV97T 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) mix and the cultures were grown for 5 days. The OD600 was measured for the starting culture and for the 5-day-old cultures using a Spectramax 250 microplate spectrophotometer system (Molecular Devices). The maximum OD600 of strain SV97T was 0·7 and was measured after 15 days at 20 °C. The experiment was repeated with three replicates at each temperature setting and net growth was calculated by subtracting the OD600 of the starting culture from the OD600 values of the 5-day-old cultures. The mean values for net growth and standard deviations were calculated.
Phylogenetic analysis of the 16S rRNA gene sequence was performed using the PHYLIP (Felsenstein, 1993) and TREEVIEW (Page, 1996) software packages 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). Phylogenetic analysis and alignment of pmoA data were performed using the software package ARB (Ludwig et al., 2004) (Fig. 2).
|
|
). Lower sequence similarity was found with members of the genus Methylosinus. Methylosinus sporium NCIMB 11126T and Methylosinus trichosporium OB3bT both showed 96 % sequence similarity to SV97T. Sequence similarity calculations indicated that the closest related nifH sequences were derived from Methylocystis echinoides IMET 10491T (97 %), ‘Methylocystis minimus’ INMI 41 (97 %) and Methylocella silvestris BL2 (91 %).
|
). Strain SV97T grew at all pH values tested, from 5·5 to 9·0, and no exospores or cysts were revealed using the methods described. Strain SV97T was obligately methane oxidizing; it did not grow on any of the alternative carbon sources tested. Strain SV97T grew well on both NMS and AMS media and grew poorly on NFMS. The presence of nifH (Poly et al., 2001) indicates that strain SV97T actively fixes nitrogen (Auman et al., 2001). A type II intracytoplasmic membrane structure, with pairwise membranes aligned parallel to the cell wall, was confirmed using transmission electron microscopy (Fig. 4). Staining with Sudan black, phase-contrast microscopy and chemical analysis indicated the presence of PHB inclusions. The major phospholipid fatty acids for strain SV97T were C18 : 18 (54·2 %), C18 : 17 (39·7 %) and C16 : 17 (6·1 %). Details of the methods used for fatty acid analysis are available as Supplementary material in IJSEM Online. Isolate SV97T had a DNA G+C content of 62 mol% (± 1 %). Strain SV97T tested negative for mmoX in PCR assays and no sMMO was detected by the colorimetric assay. Isolate SV97T had an optimum growth temperature of 27 °C, but grew well at temperatures from 14 to 37 °C (see Supplementary Fig. S1 in IJSEM Online). DNA–DNA hybridization between strain SV97T and Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T revealed 11 and 12 % DNA–DNA relatedness, respectively. Strain SV97T differs from M. parvus OBBPT in several phenotypic and genotypic characteristics; it does not have coccibacillus-shaped cells but has rod-shaped cells, does not produce cysts, has pink–red-pigmented cells, has no urease activity, has a lower DNA G+C content (62 mol%) and shows 11 % DNA–DNA hybridization and 97·2 % 16S rRNA gene sequence similarity. Strain SV97T differs from Methylocystis echinoides IMET 10491T in that it does not have coccibacillus-shaped cells, does not show spinae, has pink–red-pigmented cells, grows at pH 5·5 and 9·0, produces PHB and shows only 12 % DNA–DNA hybridization and 97 % 16S rRNA gene sequence similarity. These differences are summarized in Table 2.
|
|
|
) and which were isolated from a variety of different environments, including Baltic coast sediment, soil near an oil extraction plant and bog water (Heyer et al., 2002). From the data available (Table 1), it can be seen that none of these strains, with the exception of SV97T, are pink pigmented. One strain, Methylocystis echinoides 2 (Galchenko et al., 1977), has spinae, the other four strains have no particular distinguishing phenotype, but are all mmoX-positive. These strains may well be representatives of the same species as SV97T, but this would need to be confirmed by further characterization and DNA–DNA hybridization studies. In view of the phenotypic and genotypic differences between strain SV97T and the other Methylocystis type species (including 16S rRNA gene sequences, DNA–DNA hybridization and phenotypic characteristics), we propose that strain SV97T, isolated from wetland soil from the Svalbard Islands, Norway, represents a novel species with the name Methylocystis rosea sp. nov.
According to Rule 65 (2) of the Bacteriological Code (Lapage et al., 1992), the genus Methylocystis is incorrectly in the masculine gender. As the Greek noun kustis is in the feminine gender, the Neo-Latin name Methylocystis should also be in the feminine gender. In turn, the specific epithet of the type species, Methylocystis parvus, should correctly be parva. The specific epithet echinoides [Methylocystis echinoides (Bowman et al., 1993; Galchenko et al., 1977)] is correct, but is a Neo-Latin feminine adjective.
Description of Methylocystis rosea sp. nov.
Methylocystis rosea (ro'se.a. L. fem. adj. rosea rose-coloured, rosy).
Cells are Gram-negative, polymorphic, straight or curved fat rods, 1·1–2·5 µm long and 0·8–1·1 µm wide. Colonies on NMS agar plates are pink–red pigmented. Cells occur singly or in shapeless aggregates, but do not form rosettes when observed by light microscopy. The cells are non-motile and possess a type II intracellular membrane system. Cells do not possess sMMO, but have nifH. Cells are catalase-positive. Grows at temperatures from 5 to 37 °C. Optimal growth occurs at 27 °C, no growth occurs at 40 °C. Does not require NaCl for growth and cells are not lysed by 2 % SDS. Grows well at pH from 5·5 to 9·0. Phospholipid fatty acids are C18 : 18 (54·2 %), C18 : 17 (39·7 %) and C16 : 17 (6·1 %). No exospores or cysts have been revealed. Accumulates PHB. The DNA G+C content is 62 mol%. The type strain shows DNA–DNA hybridization values of 11 and 12%, respectively, to Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T.
The type strain, SV97T (=ATCC BAA-1196T=DSM 17261T), was isolated from a high Arctic wetland soil near Ny-Ålesund, Svalbard Islands, Norway.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
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.
Brusseau, G. A., Tsien, H. C., Hanson, R. S. & Wackett, L. P. (1990). Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity. Biodegradation 1, 19–29.[CrossRef][Medline]
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]
Dunfield, P. F., Yimga, M. T., Dedysh, S. N., Berger, U., Liesack, W. & Heyer, J. (2002). Isolation of a Methylocystis strain containing a novel pmoA-like gene. FEMS Microbiol Ecol 41, 17–26.[CrossRef]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorimetric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (1993). PHYLIP – phylogenetic interference package, version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fuse, H., Ohta, M., Takimura, O., Murakami, K., Inoue, H., Yamaoka, Y., Oclarit, J. M. & Omori, T. (1998). Oxidation of trichloroethylene and dimethyl sulfide by a marine Methylomicrobium strain containing soluble methane monooxygenase. Biosci Biotechnol Biochem 62, 1925–1931.[CrossRef][Medline]
Galchenko, V. F., Shiskina, V. N., Suzina, N. E. & Trotsenko, Y. A. (1977). Isolation and properties of new strains of obligate methanotrophs. Mikrobiologiia 46, 723–728.
Gerhardt, P. (1981). Manual of Methods for General Bacteriology. Washington, DC: American Society for Microbiology.
Graham, D. W., Korich, D. G., LeBlanc, R. P., Sinclair, N. A. & Arnold, R. G. (1992). Applications of a colorimetric plate assay for soluble methane monooxygenase activity. Appl Environ Microbiol 58, 2231–2236.
Hanson, R. S. & Hanson, T. E. (1996). Methanotrophic bacteria. Microbiol Rev 60, 439–471.
Heyer, J., Galchenko, V. F. & Dunfield, P. F. (2002). Molecular phylogeny of type II methane-oxidizing bacteria isolated from various environments. Microbiology 148, 2831–2846.
Høj, L., Olsen, R. A. & Torsvik, V. L. (2005). Archaeal communities in high Arctic wetlands at Spitsbergen, Norway (78° N) as characterized by 16S rRNA gene fingerprinting. FEMS Microbiol Ecol 53, 89–101.[CrossRef][Medline]
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D., Seeliger, H. P. R. & Clark, W. A. (editors) (1992). International Code of Nomenclature of Bacteria (1990 Revision). Bacteriological Code. Washington, DC: American Society for Microbiology.
Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.
Mandel, M., Igambi, L., Bergendahl, J., Dodson, M. L., Jr & Scheltgen, E. (1970). Correlation of melting temperature and cesium chloride buoyant density of bacterial deoxyribonucleic acid. J Bacteriol 101, 333–338.
Miguez, C. B., Bourque, D., Sealy, J. A., Greer, C. W. & Groleau, D. (1997). Detection and isolation of methanotrophic bacteria possessing soluble methane monooxygenase (sMMO) genes using the polymerase chain reaction (PCR). Microb Ecol 33, 21–31.[CrossRef][Medline]
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Poly, F., Monrozier, L. J. & Bally, R. (2001). Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol 152, 95–103.[Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Vela, G. R. & Wyss, O. (1964). Improved stain for visualization of Azotobacter encystment. J Bacteriol 87, 476–477.
Whittenbury, R., Phillips, K. C. & Wilkinson, J. F. (1970). Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61, 205–218.
Wise, M. G., McArthur, J. V. & Shimkets, L. J. (1999). Methanotroph diversity in landfill soil: isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis. Appl Environ Microbiol 65, 4887–4897.
This article has been cited by other articles:
|
|
 |
|
  A. S. Lindner, A. Pacheco, H. C. Aldrich, A. Costello Staniec, I. Uz, and D. J. Hodson Methylocystis hirsuta sp. nov., a novel methanotroph isolated from a groundwater aquifer Int J Syst Evol Microbiol, August 1, 2007; 57(8): 1891 - 1900. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
