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Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
Correspondence
Antonio Ventosa
ventosa{at}us.es
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ABSTRACT |
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Published online ahead of print on 28 January 2005 as DOI 10.1099/ijs.0.63597-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GR3T is AJ851087.
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TOPABSTRACTMAIN TEXTREFERENCES |
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The genus Methylobacterium was created to include a group of strictly aerobic, Gram-negative, rod-shaped, pink-pigmented, facultatively methylotrophic (PPFM) bacteria that can grow on one-carbon compounds such as formate, formaldehyde and methanol as the sole source of carbon and energy, as well as on a wide range of multi-carbon growth substrates (Green, 1999
). The genus Methylobacterium belongs to the 
-Proteobacteria and has the serine pathway for formaldehyde assimilation. This genus now consists of 18 species: Methylobacterium aminovorans (Urakami et al., 1993), M. aquaticum (Gallego et al., 2005), M. chloromethanicum (McDonald et al., 2001), M. dichloromethanicum (Doronina et al., 2000), M. extorquens (Bousfield & Green, 1985), M. fujisawaense (Green et al., 1988), M. hispanicum (Gallego et al., 2005), M. lusitanum (Doronina et al., 2002), M. mesophilicum (Green & Bousfield, 1983), M. nodulans (Jourand et al., 2004), M. organophilum (Patt et al., 1976), M. populi (Van Aken et al., 2004), M. radiotolerans (Green & Bousfield, 1983), M. rhodesianum (Green et al., 1988), M. rhodinum (Green & Bousfield, 1983), M. suomiense (Doronina et al., 2002), M. thiocyanatum (Wood et al., 1998) and M. zatmanii (Green et al., 1988).
The type species of the genus Methylobacterium is M. organophilum, which was the only PPFM bacterium reported to be capable of growth on methane until the description of a new methane-utilizing species, M. populi. Since M. organophilum has lost this ability, and neither the key enzyme of methanotrophic metabolism nor the genes encoding different forms of methane monooxygenase (MMO) have ever been detected in the PPFM bacteria, the methanotrophic ability of M. populi must be treated with considerable scepticism (Dedysh et al., 2004).
Members of the genus Methylobacterium are ubiquitous in nature and are thus found in a variety of habitats (Green & Bousfield, 1981, 1983), including soil, dust, freshwater and lake sediments, leaf surfaces and root nodules, rice grains, air, hospital environments and as contaminants in various products and processes. Species of Methylobacterium have been reported to exhibit resistance to chlorination (Hiraishi et al., 1995) and their presence in drinking water distribution systems is justified.
Drinking water samples were concentrated by using a tangential flow filtration system (Filtron®) and plated on Plate Count Agar (Difco) and R2A agar (Difco). Plates were incubated at 28 °C for 7 days and different morphological colonies were plated in order to obtain pure cultures. We obtained 115 isolates, 32 of which were pink-pigmented. One of these pink-pigmented pure cultures was strain GR3T, which was phenotypically characterized by using the methods described by Doronina et al. (1998). The nutritional features were determined as described by Gallego et al. (2005) by using Biolog Microplates (Biolog).
Chromosomal DNA was isolated and purified according to the methods described by Wilson (1987) and Marmur (1961) and partially modified by Hood et al. (1987). The 16S rRNA gene was amplified by using the universal primers 16F27 and 16R1488 as described by Mellado et al. (1995). Sequencing was performed by NBT-Newbiotechnic using an automated DNA sequencer (model 3100; Applied Biosystems) and an almost-complete nucleotide sequence was determined. Alignment of the 16S rRNA gene sequence was carried out with the ARB software program (Ludwig & Strunk, 1996). Phylogenetic trees were inferred by using three tree-making algorithms – maximum-parsimony, neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood. The G+C content of genomic DNA was determined by the method of Marmur & Doty (1962) and by using the equation of Owen & Hill (1979). DNA–DNA hybridization was carried out following the competition procedure of Johnson (1994), which is described in detail in Mormile et al. (1999). Hybridization temperatures were 60 and 61 °C, which are within the limit of validity for the filter method (De Ley & Tijtgat, 1970), and the percentage of hybridization was calculated according to Johnson (1994). DNA relatedness values are the mean of three values.
Strain GR3T is a Gram-negative, strictly aerobic rod that measures 1·0–1·5 µm in width by 2·0–6·0 µm in length when grown for 24 h at 28 °C. Cells are motile. Colonies of strain GR3T are circular to slightly irregular in shape, pink in colour and 2–7 mm in diameter on R2A agar (after 7 days or more); sometimes colonies can have different pink pigmentation. Strain GR3T is a slow-growing organism; no growth occurs in the presence of 1 % NaCl. Differential phenotypic characteristics of strain GR3T are summarized in Table 1.
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). The DNA G+C content of strain GR3T was 69·2 mol%, which is within the range described for the genus Methylobacterium (Green, 1999). This value is different to that of M. aquaticum (67·7 mol%) (Gallego et al., 2005). Furthermore, to determine the genotypic relatedness between strain GR3T and M. aquaticum CCM 7218T, studies of DNA–DNA hybridization were performed. DNA–DNA hybridization values obtained were 30 and 37 %, indicating that the new isolate is genotypically different from the type strain of the M. aquaticum. Low DNA–DNA hybridization values (
15 %) were also obtained between strain GR3T and other related Methylobacterium species (Table 2).
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Description of Methylobacterium variabile sp. nov.
Methylobacterium variabile (L. neut. adj. variabile variable).
Gram-negative rods, 1·0–1·5 µmx2·0–6·0 µm, occurring singly in pairs or in rosettes. Cells are motile, non-spore-forming and strictly aerobic. Colonies are pink, circular to slightly irregular and 2–7 mm in diameter after 7 days at 28 °C on R2A agar; sometimes colonies have different pink pigmentations. Slow-growing; does not grow in the presence of 1·0 % NaCl or higher. Growth occurs at 20–30 °C (optimal temperature 28 °C) and at pH 5·0–8·0 (optimal pH 6·0). Catalase- and urease-positive. Oxidase-negative. Indole, methyl red and Voges–Proskauer are negative. Starch is weakly hydrolysed. Tween 80 is hydrolysed. Gelatin, casein, aesculin and DNA are not hydrolysed. Hydrogen sulfide is not produced. Simmons' citrate test is positive. Nitrate is reduced to nitrite. Acid is produced oxidatively from D-arabinose, but not from D-glucose, D-galactose, D-mannose or maltose. Methanol, formate and formaldehyde are utilized as sole carbon sources. Ammonium sulfate, nitrate, aspartate and glutamate are utilized as sole nitrogen sources. The following compounds are utilized as sole carbon and energy sources (Biolog): D-fructose, L-fucose, D-galactose, D-gluconic acid, -D-glucose, acetic acid, -hydroxybutyric acid, 
-hydroxybutyric acid, 
-hydroxybutyric acid, -ketoglutaric acid, L-lactic acid, L-malic acid, mono-methyl succinate, propionic acid, pyruvic acid, succinamic acid, succinic acid, L-asparagine and L-glutamic acid. The following compounds are not utilized as sole carbon and energy sources (Biolog): Tween 40, Tween 80, -cyclodextrin, -cyclodextrin, dextrin, glycogen, inulin, mannan, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, amygdalin, L-arabinose, D-arabitol, arbutin, cellobiose, D-galacturonic acid, gentibiose, m-inositol, -D-lactose, lactulose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, D-melibiose, methyl -D-galactoside, 3-methyl glucose, methyl -D-glucoside, methyl -D-glucoside, methyl -D-mannoside, palatinose, D-psicose, D-raffinose, L-rhamnose, D-ribose, salicin, sedoheptulosan, D-sorbitol, stachyose, sucrose, D-tagatose, D-trehalose, turanose, xylitol, D-xylose, p-hydroxyphenyl acetic acid, -ketovaleric acid, lactamide, D-lactic acid methyl ester, D-malic acid, methyl pyruvate, alaninamide, N-acetyl-L-glutamic acid, D-alanine, L-alanine, glycyl-L-glutamic acid, L-alanyl-glycine, L-pyroglutamic acid, L-serine, putrescine, 2,3-butanediol, glycerol, adenosine, 2'-deoxyadenosine, inosine, thymidine, uridine, adenosine 5'-monophosphate, thymidine 5'-monophosphate, uridine 5'-monophosphate, fructose 6-phosphate, glucose 1-phosphate, glucose 6-phosphate and DL--glycerol phosphate.
The type strain is GR3T (=DSM 16961T=CCM 7281T=CECT 7045T), which was isolated from drinking water. The DNA G+C content of the type strain is 69·2 mol%.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMAIN TEXTREFERENCES |
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