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1 DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
2 Institut für Technische Chemie, Universität Hannover, 30167 Hannover, Germany
Correspondence
Rüdiger Pukall
rpu{at}dsmz.de
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The EMBL accession number for the 16S rDNA sequence of Paracoccus seriniphilus MBT-A4T is AJ428275.
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; Davidson et al., 2001). Also, the formation of biofilms has important implications for biochemical reactions and ecological functions in a specific ecological niche (Dagostino et al., 1991; Sternberg et al., 1999). Here, we report on the isolation and characterization of a novel bacterial strain, MBT-A4T, isolated from colonies of the North Sea bryozoan Bugula plumosa.
Isolation and morphology of strain MBT-A4T
Bryozoan samples were taken in August 1999 by divers from the south pier of the entrance harbour of the island Helgoland. Sampling depth was 7 m and samples were placed directly into polyethylene bags containing seawater from the local site. The temperature of the seawater was 17·5 °C. Samples of Bugula plumosa were washed three times for 30 min in sterile artificial sea water (Sea Salts; Sigma). For extraction of bacteria from the surface of a Bugula plumosa colony, samples (1 g) were placed into a sterile plastic bag (Seward Medical) containing 9 ml artificial seawater, and treated in a Stomacher Lab blender (Seward Medical) twice for 120 s at the highest speed. Aliquots of the resultant bacterial suspension were used as the inoculum for an enzyme screening. The L-serine basal medium used for screening experiments consisted of L-serine, 3 g l-1; Sea Salts, 34·3 g l-1; 1 ml l-1 trace element solution containing (l-1): 0·1 g ZnSO4.7H2O, 0·03 g MnCl2.4H2O, 0·3 g H3BO3, 0·2 g CoCl2.2H2O, 0·01 g CuCl2.2H2O, 0·02 g NiCl2.6H2O, 0·03 g Na2MoO4.2H2O; 1 ml l-1 vitamin solution containing (l-1): 60 mg m-inosite, 30 mg calcium pantothenate, 6·0 mg thiamin.HCl (B1); 150 µg pyridoxol.HCl (B6), 30 µg biotin. Experiments were performed in microplates (TechnoPlastic). L-Serine minimal medium (200 µl) was inoculated with 4 µl bacterial suspension extracted from Bugula plumosa. The plate was incubated at 37 °C using an Eppendorf thermal shaker. After 6 days, growth was observed in some wells, and strains able to use L-serine as their sole energy source were subcultured on L-serine basal agar medium to obtain pure cultures.
Cells of strain MBT-A4T were Gram-negative, non-spore-forming, aerobic, non-motile and catalase- and oxidase-positive. They were able to grow on marine agar (Difco), forming smooth, circular, colourless to creamy white colonies which were 1–2 mm in diameter. Strain MBT-A4T was capable of aerobic growth on L-serine as the sole carbon and nitrogen source, and produced an L-serine dehydratase (EC 4.2.1.13). This enzyme catalyses the irreversible non-oxidative deamination of L-serine to equimolar amounts of pyruvate and ammonia. The name dehydratase originates from the first partial reaction, which is a dehydration. This 
-elimination is followed by the tautomerization of the aminoacrylate and hydrolysis of the resulting imine.
Microscopy
Cells were observed by using an Olympus BH-2 microscope equipped with phase-contrast optics. Furthermore, bacteria grown on liquid broth (yeast extract/peptone medium, see Physiological characteristics) were adsorbed onto a sterile filter after reaching the mid-exponential growth phase, and fixed with 3 % glutaraldehyde in cacodylate buffer (0·1 M, pH 7·2) for 1 h at room temperature (Robinson et al., 1984). The samples were washed, dehydrated with a graded series of ethanol, critical-point-dried and sputter-coated with a gold film; samples were then examined in a Digital Scanning Microscope DSM 940 (Zeiss). Cells were cocci and short rods, 0·5–1x0·7–1 µm in size (Fig. 1) and grew as single cells or as pairs to short chains. Aggregation was observed in cultures grown in minimal medium.
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). The primer pair 27f (5'-GAGTTTGATCCTGGCTCAG-3') and 1385r (5'-CGGTGTGTRCAAGGCCC-3') was used for amplification of the 16S rRNA gene (Lane, 1991). PCR amplification of 16S rDNA was done as described previously (Pukall et al., 1999). Analysis of the 16S rDNA sequence obtained from isolate MBT-A4T followed the method described by Rainey et al. (1996), using a Taq DyeDeoxy Terminator Cycle Sequencing kit and a model 373A automated DNA sequencer (both Applied Biosystems). The sequence was manually aligned and compared with published sequences from the DSMZ 16S rDNA database, which consists of more than 6000 sequence entries, including those from the Ribosomal Database Project (Maidak et al., 2001) and EMBL. Similarity values were transformed into phylogenetic distances that compensate for multiple substitutions at any given site in the sequence (Jukes & Cantor, 1969). The algorithm of DeSoete (1983) and the neighbour-joining method contained in the PHYLIP package (Felsenstein, 1993) were used to construct phylogenetic dendrograms. All analyses were done on a SUN SparcII workstation. 16S rDNA sequence analysis indicated that strain MBT-A4T was affiliated to the genus Paracoccus within the Rhodobacter group of the 
-subclass of the Proteobacteria. The highest similarity values were 97·6, 97·4 and 97·1 % with sequences of Paracoccus marcusii, Paracoccus carotinifaciens and Paracoccus alcaliphilus, respectively, while similarities with type strains of other Paracoccus species ranged from 91·9 to 96·8 %. Dendrograms of 16S rDNA relationships obtained with the two different treeing algorithms were almost identical. The position of strain MBT-A4T relative to its phylogenetic neighbours is shown by neighbour-joining analysis (Fig. 2).
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) and determination of the DNA G+C content by HPLC (Mesbah et al., 1989) followed described procedures. DNA–DNA similarity studies were performed by using the renaturation method (Escara & Hutton, 1980; Huß et al., 1983). Hybridization conditions were as follows: 2xSSC, 10 % DMSO, 69 °C. Similarity values were calculated according to the method of Jahnke (1992). Determination of DNA relatedness between strain MBT-A4T and its closest phylogenetic relative, Paracoccus marcusii DSM 11574T, revealed a low DNA–DNA similarity of 32·6 %. The G+C content of the DNA was 63·3 mol%.
Chemotaxonomic analyses
Fatty acids were determined in cells grown in TSB medium. Fatty acid methyl esters were obtained from freeze-dried biomass (approx. 10 mg) by saponification, methylation and extraction using the modifications (Kuykendall et al., 1988) of the method of Miller (1982). The fatty acid methyl ester mixtures were separated using the 5898A Microbial Identification System (Microbial ID), which consisted of a model 3392 gas chromatograph fitted a with 5 % phenyl–methyl silicone capillary column (0·2 mmx25 m), a flame-ionization detector, a model 7673A automatic sampler and a model Kayak XA computer (all from Hewlett Packard). Peaks were integrated automatically, and fatty acid composition was determined by the identification system. The gas-chromatographic parameters were as follows: carrier gas, ultrahigh-purity hydrogen; column head pressure, 60 kPa; injection volume, 2 µl; column split ratio, 100 : 1; septum purge, 5 ml-1; column temperature, 170–270 °C at 5 °C min-1; injection port temperature, 250 °C; and detector temperature, 300 °C. Isoprenoid quinones were extracted from strain MBT-A4T grown in marine broth (Difco), as described by Collins (1985). Analysis was performed by HPLC (Groth et al., 1996) and the ubiquinone type was determined by using a QP2000 mass spectrometer fitted with a direct sample inlet device DI20 (Shimadzu) as given by Collins (1994).
The major fatty acid for strain MBT-A4T, detected by the Microbial ID system, was C18 : 1
7c (83·46 %), whereas other fatty acids were detected in smaller amounts as follows: C10 : 0 3-OH (7·92 %), C18 : 0 (2·51 %) and 11-methyl-C18 : 17c (1·48 %). This profile is characteristic of members of the -subclass of the Proteobacteria, including members of the genus Paracoccus (Kelly et al., 2000). The amounts of the major fatty acid C18 : 1 detected in Paracoccus marcusii DSM 11574T and Paracoccus carotinifaciens IFO 16121T were 79·4 and 82·0 %, respectively (Harker et al., 1998; Tsubokura et al., 1999). The major isoprenoid quinone in strain MBT-A4T was ubiquinone Q-10.
Physiological characteristics
Routinely, a complex medium containing 5 g peptone l-1, 5 g yeast extract l-1 and 34·3 g Sea Salts l-1 was used for bacterial cultivation and analysis of physiological characteristics. To induce L-serine dehydratase activity, 1 g filter-sterilized L-serine l-1 was added after autoclaving. The temperature range for growth was determined by incubating the inoculated medium (pH 7·0) at 18, 20, 25, 28, 35 and 40 °C. Growth was assessed at pH 4–11. Furthermore, the NaCl requirement was tested using 1–9 % NaCl: for this, NaCl content was adjusted by reduction of the amount of sea salts added to the medium (pH 7·0), or by addition of extra NaCl. Results were recorded after 16 h incubation.
The optimal temperature for growth was 30 °C. Growth was very poor below 20 °C and above 37 °C. Growth was observed in 1–9 % NaCl, the optimum being 3 %. Strain MBT-A4T was able to grow at pH 5–10 with an optimum range of pH 6·5–8·0. Aggregates were formed at NaCl concentrations below 2 % and at low concentrations of nutrients in minimal medium (<20 %).
The ability to utilize a variety of substrates as carbon sources was tested using the GN2 Microplates of the MicroLog system (Biolog). Bacteria were suspended in Sea Salts solution (20 g l-1), and 150 µl of this suspension was added to each well. After 24 h incubation at 30 °C, reduction of the tetrazolium dye was determined by using a Microlog plate reader. Tests for the production of arginine dihydrolase, urease, -glucosidase, protease, -galactosidase, cytochrome oxidase, fermentation of glucose, indole production from tryptophan and the reduction of nitrates to nitrites were performed using the API 20 NE system (bioMérieux) according to the manufacturer's instructions, with the exception that cells were resuspended in Sea Salts solution (34 g l-1).
Characteristics that differentiate strain MBT-A4T from other representatives of the genus Paracoccus are given in Table 1. Other properties are indicated in the species description. Strain MBT-A4T can be differentiated from its nearest phylogenetic neighbours by pigmentation, ability to reduce nitrate, lack of -glucosidase and growth on media supplemented with different carbon sources. Strain MBT-A4T also differs from P. marcusii (Harker et al., 1998) by its inability to utilize gentiobiose, arabinose, maltose, cellobiose, lactose, trehalose, furanose or sucrose, and by its ability to utilize L-serine as a carbon and energy source.
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Description of Paracoccus seriniphilus sp. nov.
Paracoccus seriniphilus (se.ri.ni'phi.lus. Gr. adj. philos loving; N.L. masc. adj. seriniphilus serine-loving).
Gram-negative, aerobic, non-motile, non-spore-forming cocci to rods, 0·5–1x0·7–1 µm in size. Colonies on marine agar are circular and colourless to creamy white. Growth occurs at 18–37 °C with an optimum of 30 °C. Grows at pH 6·5–8. NaCl is required for growth (1–9 %, optimum 3 %). Nitrate is reduced. Glucose is not fermented. No indole is produced from tryptophan. Cytochrome oxidase and -galactosidase activities are present. Arginine dihydrolase, urease and -glucosidase activities are absent. Gelatin hydrolysis is not detected. Grows on L-serine as sole carbon and nitrogen source. Cells in lag phase of growth tend to form aggregates. Utilizes Tween 40, Tween 80, N-acetyl-D-galactosamine, adonitol, D-arabitol, i-erythritol, D-fructose, D-galactose, -D-glucose, m-inositol, D-mannitol, D-mellobiose, D-sorbitol, xylitol, methyl pyruvate, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-gluconic acid, D-glucuronic acid, -hydroxybutyric acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, DL-lactic acid, propionic acid, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, L-leucine, L-ornithine, L-proline, L-pyroglutamic acid, L-serine, DL-carnitine, 
-aminobutyric acid, 2,3-butanediol and glycerol. The following substrates are not utilized: -cyclodextrin, dextrin, glycogen, N-acetyl-D-glucosamine, L-arabinose, cellobiose, L-fucose, gentiobiose, -D-lactose, lactulose, maltose, D-mannose, -methyl-D-glucose, D-psicose, D-raffinose, L-rhamnose, sucrose, D-trehalose, furanose, mono-methyl succinate, D-galacturonic acid, D-glucosaminic acid, -hydroxybutyric acid, itaconic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alanimide, L-alanylglycine, glycyl-L-aspartic acid, glycyl-L-glutamic acid, hydroxy-L-proline, L-phenylalanine, D-serine, L-threonine, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, DL--glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Ubiquinone Q-10 is the major isoprenoid quinone. In TSB medium the main fatty acid is C18 : 17c (cis-11-octadecenoic acid; cis-vaccenic acid). The DNA G+C content is 63·3 mol%.
Isolated from the North Sea bryozoan Bugula plumosa. Type strain is MBT-A4T (=DSM 14827T =CIP 107400T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Collins, M. D. (1985). Isoprenoid quinone analyses in bacterial classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. Edited by M. Goodfellow & D. E. Minnikin. London: Academic Press.
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