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Department of Microbiology and Parasitology, Faculty of Pharmacy, Universidad de 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 7 October 2005 as DOI 10.1099/ijs.0.63966-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AR27T is AM040156.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-2 subclass of the Proteobacteria and has the serine pathway for formaldehyde assimilation. It includes strictly aerobic, Gram-negative, rod-shaped, pink-pigmented, facultatively methylotrophic (PPFM) bacteria, which can grow on single 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, 2001
). At the time of writing, the genus comprises 20 recognized species (Patt et al., 1976; Green & Bousfield, 1983; Bousfield & Green, 1985; Green et al., 1988; Urakami et al., 1993; Wood et al., 1998; Doronina et al., 2000, 2002; McDonald et al., 2001; Van Aken et al., 2004; Jourand et al., 2004; Gallego et al., 2005a, b, c), with Methylobacterium organophilum as the type species (Patt et al., 1976).
Members of the genus Methylobacterium are ubiquitous in terrestrial habitats (Green & Bousfield, 1981, 1983), including soil, dust, freshwater, tap water systems, lake sediments, leaf surfaces and nodules, rice grains and air, and as contaminants in various products and processes. The PPFM bacteria are strict aerobes and can be isolated from almost any freshwater environment where some dissolved oxygen exists, e.g. from tap water systems (Gräf & Bauer, 1973) and from stratified lake systems (Hanson, 1980). Furthermore, species of this genus have been isolated from hospital environments (Gilardi & Faur, 1984), where they may pose a threat as opportunistic pathogens to seriously ill patients.
A sampling campaign in April 2004 to identify culturable and non-culturable bacteria present in the drinking water distribution system of Seville, Spain, led to isolation of a slowly growing pink-pigmented bacterium. This isolate was studied phenotypically, phylogenetically and genotypically. Based on these characteristics we suggest that strain AR27T represents a novel species of the genus Methylobacterium.
Drinking water samples (25 l) were concentrated by using a tangential flow filtration system (Filtron), plated on plate count agar (PCA; Difco) and R2A (Difco) and incubated at 28 °C for 7 days. Distinct colonies were picked in order to obtain pure cultures. Pink-pigmented colonies were selected and studied in more detail. One of these strains, AR27T, showed a characteristic growth that differentiated it from the remaining pink-pigmented colonies isolated. Strain AR27T was characterized phenotypically by using the methods described by Doronina et al. (1998). The use of different carbon compounds was determined as described previously by Gallego et al. (2005a) by using Biolog Microplates (Biolog Inc.).
Chromosomal DNA was isolated and purified according to the methods described by Wilson (1997) and a partially modified Marmur method (Marmur, 1961; Hood et al., 1987). The 16S rRNA gene was amplified using the universal primers 16F27 and 16R1488 as described by Mellado et al. (1995). Sequencing was performed by NBT-Newbiotechnic (Seville, Spain) 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 by using the ARB program (http://www.arb-home.de). Base-frequency filters were applied in the sequence comparison analysis. Phylogenetic trees were inferred by using maximum-parsimony, neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods. The G+C content of the genomic DNA was determined from the mid-point value (Tm) of the thermal denaturation profile (Marmur & Doty, 1962) by using the equation of Owen & Hill (1979).
Cells of strain AR27T were Gram-negative rods, strictly aerobic, measuring 1·0–1·2 µm wide by 1·5–5·0 µm long after 24 h growth at 28 °C. Cells were motile. Colonies of strain AR27T were circular, pink pigmented with a diameter of 0·5–0·8 mm when grown on R2A agar after 7 days incubation. Growth in liquid media was flocculant whereas on solid media growth produced colonies that usually adhered to the agar surface. Strain AR27T was slow-growing, the doubling time typically being 48–72 h under optimal conditions. Growth did not occur in the presence of 1 % NaCl. Differential phenotypic characteristics between strain AR27T and related Methylobacterium species are summarized in Table 1.
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96·4 %, with Methylobacterium fujisawaense the most similar species. Moreover, the 16S rRNA gene sequence phylogenetic analysis clearly showed that strain AR27T was located in a branch separate from the remaining species of the genus (Fig. 1). According to the phylogenetic data the isolate belongs to the genus Methylobacterium but as it only shows a relatively low similarity with other species, strain AR27T could be considered to represent a novel species.
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; Urakami et al., 1993; Green, 2001). On the basis of these molecular and physiological characteristics, we propose to place strain AR27T in a novel species, Methylobacterium adhaesivum sp. nov.
Description of Methylobacterium adhaesivum sp. nov.
Methylobacterium adhaesivum (ad.hae'si.vum. L. neut. adj. adhaesivum adhering, forming aggregates).
Cells are Gram-negative rods, 1·0–1·2x1·5–5·0 µm, occurring singly or forming aggregates (when grown in liquid R2A medium at 28 °C after 24 h). Cells are motile, non-spore-forming and strictly aerobic. Growth in liquid media is flocculant whereas growth on solid media produces colonies that usually adhere to the agar surface. Colonies are pink and circular, 0·5–0·8 mm in diameter after 7 days at 28 °C on R2A agar. Growth is slow, doubling typically requiring 48–72 h under optimal conditions. No growth in the presence of 
1·0 % NaCl. Growth occurs at 15–35 °C (optimum 28 °C) and at pH 5·0–9·0 (optimum pH 6·5). Catalase- and urease-positive. Oxidase activity is weak. Indole, methyl red and Voges–Proskauer reactions are negative. Tween, starch, gelatin, casein and DNA are not hydrolysed. Hydrogen sulfide is not produced. Simmons' citrate test is positive. Nitrate is not reduced. Acid is produced oxidatively from D-arabinose, D-glucose, D-galactose, D-mannose, fructose and 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, acetic acid, -, 
- and 
-hydroxybutyric acid, -ketoglutaric acid, L-lactic acid, D- and L-malic acid, methyl pyruvate, monomethyl succinate, propionic acid, pyruvic acid, succinamic acid, succinic acid, L-glutamic acid and glycerol. The following compounds are not utilized as sole carbon and energy sources (Biolog): Tweens 40 and 80, -cyclodextrin, -cyclodextrin, dextrin, glycogen, inulin, mannan, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, amygdalin, L-arabinose, D-arabitol, arbutin, cellobiose, L-fucose, D-galactose, D-galacturonic acid, gentiobiose, D-gluconic acid, -D-glucose, myo-inositol, -D-lactose, lactulose, maltose, maltotriose, D-mannitol, D-mannose, D-melezitose, D-melibiose, methyl -D-galactoside, 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-hydroxyphenylacetic acid, -ketovaleric acid, lactamide, D-lactic acid methyl ester, N-acetyl-L-glutamic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, glycyl L-glutamic acid, L-pyroglutamic acid, L-serine, putrescine, 2,3-butanediol, 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, AR27T (=CECT 7069T=CCM 7305T), was isolated from drinking water. The DNA G+C content of the type strain is 63·6 mol% (Tm).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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