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1 Departamento de Microbiología y Génetica, Universidad de Salamanca, Lab. 209 Edificio Departamental de Biología, Campus M. Unamuno, 37007 Salamanca, Spain
2 Cátedra de Microbiología Agrícola, Facultad de Agronomía, Universidad de Córdoba, Argentina
Correspondence
Encarna Velázquez
evp{at}gugu.usal.es
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ABSTRACT |
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-Galactosidase was produced. From 51 compounds tested 21 were used as single sources of carbon. The major respiratory lipoquinone was ubiquinone-10. The predominant cellular fatty acids were 16 : 0, 18 : 1
7c and 16 : 17c (from summed feature 3). Hydroxy fatty acids 14 : 0 2-OH and 15 : 0 iso 2-OH were present as well (from summed feature 4). The polar lipids detected in strain FA2T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and two unidentified phospholipids. The DNA G+C content of strain FA2T was 61 mol%. DNA–DNA hybridization experiments showed 27·6 % relatedness between strain FA2T and S. adhaesiva DSM 7418T. Based upon phenotypic and molecular evidence, a novel species of the genus Sphingomonas is proposed, Sphingomonas phyllosphaerae sp. nov., with strain FA2T (=LMG 21958T=CECT 5832T) as the type strain.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains FA2T and FA1 are AY453855 and AY563441, respectively.
RAPD and polar lipid data and a full phylogenetic tree are available as supplementary material in IJSEM Online.
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. Many Sphingomonas species are associated with plants and produce yellow or orange pigments (Busse et al., 2003). Recently, several novel species of this genus have been described: Sphingomonas melonis causes brown spots on yellow melon fruits (Buonaurio et al., 2002) and Sphingomonas aurantiaca, Sphingomonas areolata and Sphingomonas faenia were isolated from dust and air (Busse et al., 2003). During a study of phyllospheric bacteria of leguminous trees in central Argentina, we isolated two strains (FA1 and FA2T) from Acacia caven that produce a yellow pigment.
Strain isolation from the phyllosphere was performed according to the methods of Behrendt et al. (2003). The strains were grown on nutrient agar (Difco) for 48 h at 22 °C, developing opaque, slimy and yellow colonies. Motility was checked by phase-contrast microscopy using a Nikon HFX microscope. For electron microscopy the cells were grown in nutrient broth (Difco) for 2 days at 180 r.p.m. and at 28 °C. The samples were stained with uranyl acetate and examined at 80 kV with a Zeiss EM 209 transmission electron microscope. The strains were Gram-negative, rod-shaped and motile (1·5–1·9x0·6–0·8 µm) by means of a polar flagellum (data not shown).
Both strains were subjected to two-primer RAPD fingerprinting according to the methods of Rivas et al. (2001); they had the same pattern (available as Supplementary Fig. A in IJSEM Online), indicating that they belong to the same species.
The nearly complete 16S rRNA gene sequences (1481 nucleotides) for strains FA1 and FA2T were obtained according to the methods of Rivas et al. (2003), showing 100 % similarity, and compared with those from GenBank using BLAST (Altschul et al., 1990). This indicated that the novel strains were phylogenetically related to Sphingomonas species. 16S rRNA gene sequence similarities to all established Sphingomonas species ranged from 94·3 % (to Sphingomonas echinoides) to 97·6 % (to Sphingomonas adhaesiva). The phylogenetic tree obtained with Kimura's two-parameter correction and the neighbour-joining method (Fig. 1; a fuller phylogenetic tree is available as Supplementary Fig. B in IJSEM Online) placed strain FA2T in a separate group together with S. adhaesiva GIFU 2395T (DSM 7418T). The 16S rRNA gene sequence of strain FA2T contained the nucleotide signatures that define the genus Sphingomonas according to Takeuchi et al. (2001).
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. Supplementary API 20NE and API 50CH galleries (bioMérieux) were used for characterization. The novel strains had the same phenotypic characteristics. Differential phenotypic characteristics between these strains and the phylogenetically closest species (Sphingomonas sensu stricto) are shown in Table 1. The strains isolated from rhizosphere of Acacia caven differ from S. adhaesiva, the most closely related species, in gelatinase production, acid production from glycerol, salicin and galactose and assimilation of mannose, maltose and citrate.
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7c and 16 : 17c (from summed feature 3). The hydroxy fatty acids detected were 14 : 0 2-OH and 15 : 0 iso 2-OH (from summed feature 4). The fatty acid profile of strain FA2T is characteristic of strains from Sphingomonas sensu stricto (Kämpfer et al., 1997; Takeuchi et al., 2001). Main differences between strain FA2T and S. adhaesiva are the presence of 14 : 0, 15 : 0 and 11-methyl 18 : 17c, which were not detected in S. adhaesiva (Kämpfer et al., 1997; Takeuchi et al., 2001). Quantitative differences in the non-polar saturated fatty acid 16 : 0 and in the unsaturated acid 17 : 16c were observed.
Quinone and lipid compositions were determined by HPLC (Tindall, 1990). Similarly to other Sphingomonas species (Busse et al., 1999; Takeuchi et al., 2001; Yabuuchi et al., 2002), the major respiratory lipoquinone in strain FA2T was ubiquinone Q-10. The polar lipid pattern of strain FA2T after separation by two-dimensional TLC is available as Supplementary Fig. C. The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid and two unidentified phospholipids, PL1 and PL2. Phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine were detected in small amounts. This polar lipid pattern is in good agreement with those found in other Sphingomonas species (Busse et al., 1999), especially with that of S. adhaesiva according to the data of Kämpfer et al. (1997). The two unidentified phospholipids in FA2T have only been detected infrequently in a few Sphingomonas species (Busse et al., 1999).
DNA G+C content was determined according to the methods of Chun & Goodfellow (1995); the G+C content of strain FA2T was 61 mol%, which is similar to other Sphingomonas species (Yabuuchi et al., 2002). DNA–DNA hybridization analyses were done as described by Huß et al. (1983) and Escara & Hutton (1980). Renaturation rates were computed with the TRANSFER.BAS software by Jahnke (1992). DNA–DNA relatedness was tested in 2xSSC plus 10 % (v/v) DMSO at 68 °C. The results of DNA–DNA hybridization showed 100 % relatedness between strains FA1 and FA2T and 27·6 % between strain FA2T and S. adhaesiva DSM 7418T, indicating that the novel strains do not belong to S. adhaesiva when a threshold value of 70 % DNA–DNA relatedness is adopted for defining species (Wayne et al., 1987). On the basis of phylogenetic, chemotaxonomic and phenotypic data, we propose that strain FA2T classified as the type strain of Sphingomonas phyllosphaerae sp. nov.
Description of Sphingomonas phyllosphaerae sp. nov.
Sphingomonas phyllosphaerae (phyl.lo.sphaer'ae. N.L. gen. fem. n. phyllosphaerae of the phyllosphere).
Gram-negative, strictly aerobic, non-spore-forming, rod-shaped cells, 1·5–1·9 µm long and 0·6–0·8 µm in diameter. Motile by polar flagellation. Colonies on nutrient agar are circular convex, slimy, yellow, opaque and usually 1–2 mm in diameter within 2 days of growth at 25 °C. Major respiratory lipoquinone is ubiquinone Q-10. Major non-polar fatty acids detected in the type strain are 16 : 0, 18 : 17c and 16 : 17c (from summed feature 4). Hydroxy fatty acids detected are 14 : 0 2-OH and 15 : 0 iso 2-OH (from summed feature 4). Polar lipids detected in the type strain are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid and two unidentified phospholipids (PL1 and PL2). Oxidizes glucose in media containing ammonium nitrate as nitrogen source, but is unable to ferment glucose in the same media. Produces catalase and -galactosidase, and oxidase weakly, but not gelatinase, urease, arginine dehydrolase, tryptophan deaminase, indol or H2S. Aesculin is hydrolysed. Utilizes L-arabinose, D-xylose, galactose, D-fructose, D-fucose, melibiose, methyl 
-D-glucoside, L-rhamnose, amygdalin, arbutin, cellobiose, lactose, sucrose, trehalose, D-raffinose, N-acetylglucosamine, turanose, adipate and malate as sole carbon sources. Does not grow in D-arabinose, L-xylose, ribose, L-sorbose, L-lyxose, L-fucose, inositol, mannitol, adonitol, glycerol, D-arabitol, L-arabitol, xylitol, caprate, gluconate, 2-ketogluconate, citrate, methyl -xyloside, methyl -D-mannoside, salicin, inulin, melezitose, starch, glycogen, erythritol, sorbitol, dulcitol, maltose, -gentiobiose, D-tagatose, 5-ketogluconate or phenylacetate.
Strains FA1 and FA2T were isolated from Acacia caven phyllosphere. The type strain FA2T (=LMG 21958T=CECT 5832T) has a DNA G+C content of 61 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Behrendt, U., Ulrich, A. & Schumann, P. (2003). Fluorescent pseudomonads associated with the phyllosphere of grasses; Pseudomonas trivialis sp. nov., Pseudomonas poae sp. nov. and Pseudomonas congelans sp. nov. Int J Syst Evol Microbiol 53, 1461–1469.
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55 %. Bar, 1 nt substitution per 100 nt. Erythrobacter longus was used as the outgroup.