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1 G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia
2 Bioengineering Center, Russian Academy of Sciences, 117312 Moscow, Russia
3 Institute of Microbiology, Russian Academy of Sciences, 117312 Moscow, Russia
4 Department of Applied Chemistry and Microbiology, University of Helsinki, PO Box 56, FIN 00014, Finland
Correspondence
Yuri A. Trotsenko
trotsenko{at}ibpm.pushchino.ru
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Published online ahead of print on 27 February 2004 as DOI 10.1099/ijs.0.02956-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain F31T is AY298905.
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; Urakami & Komagata, 1986), Methylophilus (Jenkins et al., 1987) and Methylovorus (Govorukhina & Trotsenko, 1991). Marine obligate methylobacteria assigned to the genus Methylophaga (Urakami & Komagata, 1987) are clearly distinguished from members of the genera Methylobacillus, Methylophilus and Methylovorus by their tolerance to NaCl (up to 10–12%, w/v), some physiological characteristics and the low DNA G+C content (38·0–49·0 mol%). However, terrestrial and marine strains of obligate methylobacteria possess similar morphology and metabolic organization. Thus, the main criteria used to classify obligate methylobacteria into separate genera and species are their genomic and phylogenetic characteristics. Here, we present a formal taxonomic description of the newly isolated, obligately methylotrophic strain F31T, which has the RuMP pathway. The name Methylobacillus pratensis sp. nov. is proposed for this isolate.
Strain F31T was isolated from meadow grass (Poa trivialis L.) sampled from the city park in Helsinki (Finland) and grown on medium K (Doronina et al., 1998). Analyses of phenotypic and genotypic properties of the novel isolate were performed as described previously (Doronina et al., 2003). The 16S rRNA gene of strain F31T was amplified by PCR, sequenced and screened against sequences within the GenBank database by using BLAST (http://www.ncbi.nlm.nih.gov/blast). The 16S rRNA gene sequence of strain F31T was then aligned with a representative set of 16S rRNA gene sequences obtained from recent GenBank releases, using CLUSTAL W software (Thompson et al., 1994). Positions of sequence and alignment uncertainty were omitted, and a total of 1308 nucleotides was used in the phylogenetic analysis. Phylogenetic trees were constructed by using various algorithms implemented in TREECON (Van de Peer & De Wachter, 1994).
Cells of strain F31T are Gram-negative, asporogenous rods (0·5–0·7x0·9–1·8 µm) that are motile by means of a polar flagellum, and occur singly or (rarely) in pairs (Fig. 1a–c). Reproduction occurs by binary fission.
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, the dominant cellular fatty acids of strain F31T are unsaturated C16 : 1
7 acid (46·6 %) and straight-chain saturated C16 : 0 acid (41·6 %). The presence of 3-hydroxy fatty acids was observed, but no 2-hydroxy fatty acids were found. The data indicate a considerable similarity in the fatty acid composition of strain F31T and the type cultures of the genus Methylobacillus, i.e. Methylobacillus glycogenes ATCC 29475T and Methylobacillus flagellatus DSM 6875T. Analysis of the cellular phospholipid composition revealed the presence of phosphatidylethanolamine (65 %), phosphatidylglycerol (20 %), diphosphatidylglycerol (cardiolipin) (10 %) and minor amounts of phosphatidylserine, phosphatidic acid and an unidentified phospholipid. Phosphatidylcholine and lysolecithin were not found.
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). Methylamine dehydrogenase PQQ and amine oxidase were absent in methylamine-grown cells. Alternatively, the isolate possessed an inducible 
-glutamylmethylamide synthetase and N-methylglutamate synthase/lyase, the specific enzymes of the N-methylglutamate pathway producing formaldehyde, which is further oxidized by glutathione-dependent formaldehyde dehydrogenase to formate. The latter is partly oxidized to CO2 by NAD-dependent formate dehydrogenase. Formaldehyde assimilation occurs via the RuMP cycle (Entner–Doudoroff variant), as confirmed by the presence of 3-hexulose phosphate synthase and 2-keto-3-deoxy-6-phosphogluconate aldolase. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are active with both NAD+ and NADP+. Rather high levels of these enzymes indicate the preferential oxidation of formaldehyde to CO2 via the dissimilatory hexulose phosphate cycle, which provides the methylotroph with the reduced equivalents and energy for biosynthesis. However, we cannot rule out the same potential role of the tetrahydromethanopterin (H4MPT)-dependent oxidation pathway in formaldehyde dissimilation in our isolate because high activities of methenyl H4MPT cyclohydrolase and NAD(P)-dependent methylene H4MPT dehydrogenases were found in Methylobacillus flagellatus KTT (Vorholt et al., 1999).
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The tricarboxylic acid cycle is deficient in 2-oxoglutarate dehydrogenase. The absence of isocitrate lyase and malate synthase indicates the non-functional glyoxylate shunt in strain F31T. Oxaloacetate is replenished by phosphoenolpyruvate carboxylase. Primary ammonia assimilation occurs by reductive amination of 2-oxoglutarate to glutamate, since the glutamine synthetase/glutamate synthase pathway (GS/GOGAT) is not operative in this organism.
In the phylogenetic tree derived from 16S rRNA gene sequences (Fig. 2), strain F31T consistently branched with the 
-Proteobacteria. The relatively high level of 16S rRNA gene sequence similarity (95–97 %) between the strain and members of the genus Methylobacillus indicated a close relationship. The level of DNA relatedness between the novel isolate and reference strains of the genus Methylobacillus (Methylobacillus glycogenes ATCC 29475T and Methylobacillus flagellatus DSM 6875T) was in the range 28–34 %. Remarkably, strain F31T had a very low degree of DNA hybridization (5–10 %) with members of the genera Methylophilus, Methylovorus and Methylophaga (Methylophilus methylotrophus NCIMB 10515T, Methylovorus glucosotrophus ATCC 49758T and Methylophaga marina ATCC 35842T). Consequently, strain F31T is classified as the type strain of a novel Methylobacillus species, for which the name Methylobacillus pratensis sp. nov. is proposed (Table 3).
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Gram-negative rods that are 0·9–1·8x0·5–0·7 µm in size. Multiply by binary fission. Cells are motile by means of a single polar flagellum. Colonies on mineral salts/methanol agar are white and 1–2 mm in diameter. Strictly aerobic. Growth factors not required. Able to grow at 10–37 °C, at pH 5·5–8·5 and optimally at 25–30 °C and pH 6·5–7·5. Urease-, catalase- and oxidase-positive. Nitrate is reduced to nitrite. Produces indole (indole 3-acetic acid) from tryptophan on medium with nitrate as nitrogen source. No growth occurs in the presence of 3 % (w/v) NaCl. Obligate methylotroph that utilizes only methanol and methylamine. Methylamine is oxidized to formaldehyde by the N-methylglutamate pathway enzymes, -glutamylmethylamide synthetase and N-methylglutamate synthase/lyase. Formaldehyde is assimilated via the RuMP pathway (Entner–Doudoroff variant). Ammonia is assimilated by glutamate dehydrogenase. Tricarboxylic acid cycle is incomplete at the level of 2-oxoglutarate dehydrogenase; the glyoxylate shunt enzymes are absent. Nitrates, ammonium salts, methylamine, glutamate and urea serve as nitrogen sources. The prevailing cellular fatty acids are C16 : 0 and C16 : 1. The major ubiquinone is Q-8. Dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol (cardiolipin). Produces exopolysaccharide containing glucose, galactose and xylose. DNA G+C content is 61·5 mol% (Tm).
The type strain is F31T (=VKM B-2247T=NCIMB 13994T), which was isolated from meadow grass (Poa trivialis L.) growing in Helsinki (Finland).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Doronina, N. V., Darmaeva, T. D. & Trotsenko, Y. A. (2003). Methylophaga alcalica sp. nov., a novel alkaliphilic and moderately halophilic, obligately methylotrophic bacterium from an East Mongolian saline soda lake. Int J Syst Evol Microbiol 53, 223–229.
Govorukhina, N. I. & Trotsenko, Y. A. (1991). Methylovorus, a new genus of restricted facultatively methylotrophic bacteria. Int J Syst Bacteriol 41, 158–162.
Jenkins, O., Byrom, D. & Jones, D. (1987). Methylophilus: a new genus of methanol-utilizing bacteria. Int J Syst Bacteriol 37, 446–448.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Urakami, T. & Komagata, K. (1986). Emendation of Methylobacillus Yordy and Weaver 1977, a genus for methanol-utilizing bacteria. Int J Syst Bacteriol 36, 502–511.
Urakami, T. & Komagata, K. (1987). Characterization of species of marine methylotrophs of the genus Methylophaga. Int J Syst Bacteriol 37, 402–406.
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Vorholt, J. A., Chistoserdova, L., Stolyar, S. M., Thauer, R. K. & Lidstrom, M. E. (1999). Distribution of tetrahydromethanopterin-dependent enzymes in methylotrophic bacteria and phylogeny of methenyl tetrahydromethanopterin cyclohydrolases. J Bacteriol 181, 5750–5757.
Yordy, J. R. & Weaver, T. Y. (1977). Methylobacillus: a new genus of obligate methylotrophic bacteria. Int J Syst Bacteriol 27, 247–255.
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