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1 Eötvös Loránd University, Faculty of Science, Department of Microbiology, Pázmány Péter sétány 1/c, H-1117 Budapest, Hungary
2 DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, D-38124 Braunschweig, Germany
Correspondence
Erika M. Tóth
totherika{at}ludens.elte.hu
| ABSTRACT |
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| MAIN TEXT |
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Strain D287T was isolated in August 2004 from a whole-body homogenate sample of Daphnia cucullata (Crustacea: Cladocera) collected from Lake Balaton at Tihany (4 ° 55' 20'' N 1 ° 55' 39'' E), Hungary: 30 adult individuals of D. cucullata were selected under a stereomicroscope (Leica), washed five times with 5 ml sterile water from the lake, homogenized with a sterile glass mortar and used as composite sample. Bacterial strain D287T was isolated and later maintained on King B agar medium (King et al., 1954
).
Colony morphology was studied on King B agar medium by direct and stereomicroscopic observations of single colonies. Cell morphology and motility were studied by native preparations and by Gram staining according to Claus (1992)
.
Growth under anaerobic conditions was examined by using slant agar cultures on King B medium incubated in an anaerobic chamber (Forma Scientific). Oxidase activity was tested according to the method of Tarrand & Gröschel (1982)
. Catalase production and the Voges–Proskauer reaction were investigated by using the methods of Cowan & Steel (1974)
. Acid production from glucose was studied by the O/F test according to Hugh & Leifson (1953)
. Growth at different temperatures (4, 28, 38, 45 °C), NaCl concentration (0, 2.5, 5, 10 %, w/v) and pH (pH 4–12 in steps of 0.5 pH units) were determined by using King B agar or broth. Urease activity, nitrate reduction, starch hydrolysis, indole production from tryptophan, caseinase, gelatinase and phosphatase activities, hydrolysis of Tween 80 and H2S production from cysteine were studied according to the methods of Smibert & Krieg (1994)
. Haemolysin production was tested on ox-blood agar plates (Elek & Levy, 1954
). Chitinase activity was studied according to a modified version of the method of Holding & Collee (1971)
. Other enzyme activities were tested by using the API ZYM system (bioMérieux). Utilization of 95 different carbon sources as sole source of carbon was studied on Biolog GP2 microplates by using an ELISA reader (SUNRISE Remote; Tecan). System 3.5 was used to evaluate the results according to the manufacturer's instructions. For comparison, Biolog test results were evaluated with N. alkalitolerans DSM 16699T, N. dubius DSM 19084T and N. jensenii DSM 20641T.
The cell-wall diamino acid of strain D287T was determined from whole-cell hydrolysates as described by Hasegawa et al. (1983)
. Isoprenoid quinones were extracted from cells cultivated in liquid Rich medium (Yamada & Komagata, 1972
) according to the method of Collins et al. (1977)
and the profile was analysed by HPLC (HP 9001; Hewlett Packard) (Groth et al., 1997
). Cellular fatty acids were extracted from cells cultivated on trypticase soy agar (Difco) at 28 °C according to Stead et al. (1992)
and were analysed by GC (Groth et al., 1996
). Polar lipids were determined according to the method described by Minnikin et al. (1979)
. The DNA base composition was determined after disruption of bacterial cells by using a French press. After purification on hydroxyapatite following the procedure of Cashion et al. (1977)
, the DNA was degraded to nucleosides by using P1 nuclease and bovine intestinal mucosa alkaline phosphatase as described by Mesbah et al. (1989)
. The nucleosides were separated by reversed-phase HPLC (HP 9001) according to Tamaoka & Komagata (1984)
. The G+C content of the DNA was calculated from the ratio of deoxyguanosine to thymidine.
Extraction of genomic DNA and PCR-mediated amplification of the 16S rRNA gene of strain D287T were carried out by using the methods of Rainey et al. (1996)
. The PCR product was purified by using a Prep-A-Gene kit (Bio-Rad). Cycle sequencing was performed with the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's protocol, and sequence analysis was performed in an Applied Biosystems model 310 Genetic Analyzer.
A BLAST (Altschul et al., 1997
) and GenBank database search (http://www.ncbi.nlm.nih.gov/) for 16S rRNA gene sequences similar to that of strain D287T was performed. Pairwise similarity values to the phylogenetically closest type strains of recognized members of the genus Nocardioides were calculated by using the SimTable analysis of the EzTaxon server (http://www.eztaxon.org). The 16S rRNA gene sequence of strain D287T was manually aligned by using the BioEdit program (Hall, 1999
) against sequences available from EMBL. Evolutionary distances were calculated according to the Kimura two-parameter method (Kimura, 1980
) and a phylogenetic dendrogram was constructed by using the neighbour-joining method (Saitou & Nei, 1987
) within the CLUSTAL_X software package (Thompson et al., 1997
). Bootstrap analysis was based on 1000 resamplings (Felsenstein, 1985
).
Morphological, cultural and physiological characteristics of strain D287T are given in the species description below. Phenotypic characteristics that differentiate strain D287T from its closest phylogenetic neighbours are given in Table 1
.
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9c (9.88 %), C17 : 0 10-methyl (9.67 %), iso- C17 : 1
9c (8.10 %), iso-C17 : 0 (8.09 %), C17 : 1
8c (3.55 %), iso-C16 : 1 (2.63 %), anteiso-C17 : 0 (2.44 %), iso-C18 : 0 (2.35 %), C16 : 1
7c (2.19 %), iso-C14 : 0 (1.72 %), iso-C15 : 0 (1.64 %) and C18 : 1
7c (1.15 %). This fatty acid profile, with a high proportion of iso- and anteiso-branched chain fatty acids, clearly differentiated strain D287T from Marmoricola aurantiacus, which has a cellular fatty acid pattern with straight-chain saturated and monounsaturated components and 10-methyl octadecanoic (tuberculostearic) acid as the only branched-chain fatty acid (Urzì et al., 2000
Strain D287T showed 16S rRNA gene sequence similarity values below 97 % to the type strains of all recognized species of the genus Nocardioides, the value generally accepted for the delineation of separate genomic species (Stackebrandt & Goebel, 1994
; Stackebrandt & Ebers, 2006
). It could additionally be differentiated from its phylogenetically nearest neighbours on the basis of several phenotypic characteristics (Table 1
). It is therefore concluded that strain D287T represents a novel species of the genus Nocardioides, for which the name Nocardioides daphniae sp. nov. is proposed.
Description of Nocardioides daphniae sp. nov.
Nocardioides daphniae (daph'ni.ae. N.L. gen. n. daphniae pertaining to the water flea genus Daphnia, as the type strain was isolated from Daphnia cucullata).
Gram-positive, aerobic, oxidase-negative, catalase-positive bacterium. Cells are non-motile, short rods or coccoids (0.8–1.0x1.2–2.2 µm). Colonies on King B agar medium are circular, convex, smooth, glistening and of yellowish colour. Spores and mycelia are not formed. The temperature range for growth is 4–38 °C, with optimum growth at 28 °C. The pH range for growth is pH 5.5–10.5, with optimum growth between pH 7 and 9. Growth occurs in the presence of 0–5 % NaCl. Good growth occurs on King B and nutrient agar. H2S and indole are not produced. Negative for the Voges–Proskauer and
-glucosidase reactions. Acid is not produced from glucose. Haemolysin is not detected. Positive for production of chitinase. Weakly positive for caseinase. D-Fructose,
-D-glucose, D-mannose, trehalose, D-ribose, methyl β-D-glucoside, palatinose, psicose, sorbitol, stachyose, acetic acid, β-hydroxybutyric acid,
-hydroxybutyric acid,
-ketovaleric acid, L-lactic acid, D-malic acid, methylpyruvate, glycyl L-glutamic acid, pyruvic acid, 2,3-butanediol, adenosine, 2-deoxyadenosine, inosine, thymidine, uridine, adenosine 5'-monophosphate and uridine 5'-monophosphate are utilized as sole carbon source.
-Cyclodextrin, β-cyclodextrin, dextrin, glycogen, inulin, mannan, Tweens 40 and 80, N-acetylglucosamine, L-arabinose, arabitol, arbutin, D-cellobiose, D-galactose, myo-inositol, maltose, D-melezitose, D-melibiose, D-mannitol, D-raffinose, L-rhamnose, sucrose, D-xylose, L-pyroglutamic acid, glycerol, D-galacturonic acid, gentiobiose, D-gluconic acid, lactulose, maltotriose, methyl
-D-galactoside, methyl β-D-galactoside, methyl
-D-mannoside, salicin, sedoheptulosan, tagatose, xylitol,
-hydroxybutyric acid, p-hydroxyphenylacetic acid, lactamide, D-lactic acid methyl ester, methyl
-D-glucoside,
-D-lactose, L-malic acid, monomethyl succinate, succinamic acid, succinic acid, N-acetyl-L-glutamic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-serine, putrescine, N-acetyl-D-mannosamine, amygdalin, fucose, 3-methyl glucose, turanose,
-ketoglutaric acid, propionic acid, L-glutamic acid, thymidine 5'-monophosphate, DL-
-glycerol phosphate, D-glucose 6-phosphate, D-fructose 6-phosphate, and glucose 1-phosphate are not utilized. The diagnostic diamino acid of the peptidoglycan is LL-2,6 diaminopimelic acid. The major menaquinone is MK-8(H4). Phosphatidylglycerol and diphosphatidylglycerol occur as polar lipids. The predominant cellular fatty acid is iso-C16 : 0. The DNA G+C content is 69.9 mol%. Other phenotypic characteristics are given in Table 1
.
The type strain, D287T (=DSM 18664T=CCM 7403T), was isolated from the water flea Daphnia cucullata originating from Lake Balaton in Hungary.
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