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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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) and R. gilardii subsp. rosea (Han et al., 2003), R. cervicalis, R. fauriae (Rihs et al., 1993), R. mucosa (Han et al., 2003) and R. lacus (Jiang et al., 2006). Members of the genus Roseomonas belong to the Alphaproteobacteria and are pink, Gram-negative coccobacilli with oxidative metabolism that, historically, have been cultured from clinical specimens and are considered to be pathogenic for humans (Rihs et al., 1993; McLean et al., 2006). The recent description of R. lacus (Jiang et al., 2006), isolated from freshwater sediment, proves that species of this genus can be isolated from environmental samples. Moreover, other bacterial strains related to this genus have been isolated from very different environments such as drinking water distribution systems (September et al., 2004) or associated with deep-water marine invertebrates (Sfanos et al., 2005), indicating that members of the genus Roseomonas are widely distributed in nature (Jiang et al., 2006).
During a broad study carried out in 2003–2004 that focussed on the identification of culturable bacteria occurring in the drinking water distribution system of Seville, Spain, approximately 600 bacterial strains were isolated. Drinking water samples (25 l) were concentrated 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. Morphologically different colonies were picked in order to obtain pure cultures. The majority of the pink-pigmented isolates belonged to the genus Methylobacterium (Gallego et al., 2005a, b, c, 2006), but strain TR53T differed strongly from isolates related to this genus, being unable to grow on one-carbon compounds such as methanol. This isolate was studied phenotypically, phylogenetically and genotypically.
Chromosomal DNA was isolated and purified according to the method described by Marmur (1961). The 16S rRNA gene was amplified using universal primers 16F27 and 16R1488 as described by Mellado et al. (1995). The almost-complete nucleotide sequence was determined by NBT-Newbiotechnic (Seville, Spain) using an automated DNA sequencer model 3100 (Applied Biosystems). Subsequent sequence analysis was conducted using the ARB program package (Ludwig et al., 2004). Following the recommendations of Ludwig et al. (1998), alternative treeing methods (maximum-parsimony, distance matrix and maximum-likelihood) were used. A comparison of 16S rRNA gene sequences revealed that the sequence of strain TR53T (1387 bp) displayed the highest level of similarity to those of Roseomonas species; highest sequence similarities were to Roseomonas genomospecies 5 strain ATCC 49960 (95.7 %) and R. cervicalis ATCC 49957T (94.9 %). The sequence similarity between strain TR53T and R. fauriae ATCC 49958T was only 85.6 %. The similarity values between R. fauriae and Roseomonas species with validly published names were lower than 86.3 %, indicating that R. fauriae probably represents a member of a different genus, as has been suggested previously (Cohen et al., 2004; Han et al., 2003; Jiang et al., 2006). Similarly, the similarity between Roseomonas genomospecies 6 and R. fauriae is 96.6 % and that between Roseomonas genomospecies 6 and the other Roseomonas species is lower than 86.2 %. Overall, the 16S rRNA phylogenetic analysis clearly shows that strain TR53T constitutes a branch of its own that is separate from the other species of the genus Roseomonas (Fig. 1). Our results suggest that the proposal of the genera Teichococcus and Muricoccus should be investigated in detail since they might represent species of the genus Roseomonas. However, this genus is rather heterogeneous and more in-depth studies are necessary. On the other hand, the similarity between strain TR53T and the type strains of species of the genera Teichococcus and Muricoccus were 94.9 and 95.5 %, respectively. According to phylogenetic data, strain TR53T belongs to the genus Roseomonas, but as it only shows a relatively low similarity to other species, strain TR53T represents a novel species of this genus.
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) using the equation of Owen & Hill (1979), as described previously (Ventosa et al., 1999). The DNA G+C content of strain TR53T is 68.6 mol%, a value that is within the range for members of the genus Roseomonas (Rihs et al., 1993; Jiang et al., 2006).
The shape and motility of bacterial cells were observed under a phase-contrast microscope (x1000) from a 48 h liquid culture in R2A medium. Growth at different temperatures (4–40 °C), pH (4–12) and NaCl concentrations (0–5 % NaCl) was tested on R2A agar medium. The isolate was also tested for its ability to grow on nutrient agar medium (NM) and tryptone soy agar (TSA; Difco). H2S production and acid and gas production from glucose, lactose and sucrose were determined on Kligler iron agar (Difco). Oxidase activity was detected using a 1 % solution of tetramethyl-p-phenylenediamine (Difco) (Kovacs, 1956). Catalase activity was tested by picking up a young colony and smearing it in a drop of H2O2. The methyl red and Voges–Proskauer reactions were performed in Clark–Lubs' medium (Scharlau). Indole production was determined with Kovacs' reagent in 1 % tryptone broth. Simmons' citrate test was carried out in Simmons' citrate agar (Sigma). For the determination of acid production from different carbohydrates (1 %), a medium containing 0.5 % peptone, 0.5 % NaCl and 0.001 % phenol red was used (Cowan & Steel, 1974). Nitrate reduction was tested on nitrate broth containing 0.2 % KNO3 (Skerman, 1967). Urease activity was studied in Christensen's medium (Christensen, 1946). Hydrolysis of gelatin, starch and DNA was tested in the corresponding agar media (Scharlau). Tween 80 hydrolysis was tested in R2A medium containing 1 % Tween 80 and 0.02 % CaCl2. Casein hydrolysis was tested in R2A medium supplemented with 2 % skimmed milk (Difco). Use of methanol was tested in mineral medium K (Doronina et al., 1998). API 20NE and API ZYM strips (bioMérieux) were inoculated according to the manufacturer's instructions and incubated at 28 °C. API 50CH was inoculated as described by Kersters et al. (1984). API ZYM strips were read after 4 and 24 h and API 20NE and API 50CH were read after 7 and 14 days. Antibiotic susceptibility was determined according to the conventional Kirby–Bauer method (Bauer et al., 1966).
Strain TR53T is a Gram-negative coccobacillus that is strictly aerobic and measures 1.0 µm wide by 1.2–2.0 µm long when grown for 48 h at 28 °C in R2A liquid medium. Cells were non-motile. Colonies of strain TR53T were irregular, pale-pink-pigmented and mucoid with a diameter of 0.5–1.0 mm on R2A agar after 5 days incubation at 28 °C. On NM, colonies were not mucoid. Strain TR53T was slow-growing, typically requiring 3–4 days under optimal conditions to give observable turbidity. Growth did not occur in the presence of 
2 % NaCl. Other phenotypic features are shown in the species description. Differential phenotypic characteristics of strain TR53T and other Roseomonas species are summarized in Table 1.
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; Kämpfer & Kroppenstedt, 1996). The predominant fatty acids of strain TR53T were C18 : 1
7c, iso-C15 : 0 2-OH and/or C16 : 17c, C18 : 1 2-OH and C16 : 0. This is similar to the fatty acid profile of R. gilardii subsp. rosea (Han et al., 2003), except that strain TR53T has a higher percentage of iso-C15 : 0 2-OH and/or C16 : 17c. However, the fatty acid profile of strain TR53T differed from that reported for R. lacus (Jiang et al., 2006). Analysis of quinones was carried out by the Identification Service of the DSMZ (Braunschweig, Germany). As with other member of the genus Roseomonas, the major quinone is ubiquinone-10 (Q-10). On the basis of the molecular and physiological characteristics of strain TR53T, it is proposed that this strain represents a novel species, Roseomonas aquatica sp. nov.
Description of Roseomonas aquatica sp. nov.
Roseomonas aquatica (a.qua'ti.ca. L. fem. adj. aquatica found in water, aquatic).
Gram-negative coccobacillus, 1.0x1.2–2.0 µm, occurring singly and in pairs (in liquid R2A medium at 28 °C after 48 h) (Fig. 2). Cells are non-motile, non-spore-forming and strictly aerobic. Colonies are irregular, pale-pink-pigmented, mucoid, with a diameter of 0.5–1.0 mm on R2A agar after 4 days incubation at 28 °C. On NM, colonies are not mucoid and have a diameter 
0.5 mm after 4 days incubation. Slow-growing, typically requiring 2–4 days under optimal conditions in order to observe turbidity in liquid media. Does not grow on TSA. Does not grow in the presence of 2 % NaCl. Growth occurs at 15–35 °C (optimal temperature 25–28 °C) and pH 5.0–9.0 (optimal pH 7.0). Catalase- and urease-positive. Oxidase-negative. Indole, methyl red and Voges–Proskauer reactions and Simmons' citrate test are negative. Tween 80, starch, gelatin, casein and DNA are not hydrolysed. Hydrogen sulfide is not produced. Nitrate is reduced to nitrite. Acid is not produced oxidatively from D-galactose, D-mannose, D-glucose, D-fructose, D-maltose, glycerol, D-mannitol, D-trehalose, D-xylose or lactose. Does not use any of the carbohydrates or acids present in API 20NE and API 50CH strips as sole carbon sources, i.e. glycerol, erythritol, D- and L-arabinose, D-ribose, D- and L-xylose, D-adonitol, methyl 
-D-xylopyranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl 
-D-mannopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, D-cellobiose, D-maltose, D-lactose, D-melibiose, sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- and L-fucose, D- and L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase are present. Negative for arginine dihydrolase, lipase (C14), valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Cellular fatty acids are C12 : 0 (0.6 %), iso-C16 : 0 and/or C14 : 0 3-OH (2.7 %), iso-C15 : 0 2-OH and/or C16 : 17c (27.0 %), C16 : 0 (12.5 %), C16 : 0 3-OH (2.9 %), C18 : 17c (31.3 %), C18 : 0 (1.5 %) and C18 : 1 2-OH (21.5 %). The major quinone is ubiquinone-10 (Q-10). Resistant to (µg per disc) tetracycline (30), rifampicin (30), streptomycin (10), neomycin (10), erythromycin (15), kanamycin (30), vancomycin (30), nalidixic acid (30), novobiocin (30), penicillin (10 units), bacitracin (10 units), cephalothin (30), chloramphenicol (30) and trimethoprim/sulfamethoxazole (1.25/23.75).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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