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Int J Syst Evol Microbiol 56 (2006), 2387-2390; DOI  10.1099/ijs.0.64275-0
© 2006 International Union of Microbiological Societies

Paracoccus homiensis sp. nov., isolated from a sea-sand sample

Byung-Yong Kim1, Hang-Yeon Weon2, Seung-Hee Yoo1, Soon-Wo Kwon1, Yang-Hee Cho1, Erko Stackebrandt3 and Seung-Joo Go1

1 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, Rural Development Administration (RDA), Suwon 441-707, South Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, South Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr


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Strain DD-R11T, isolated from a sea-sand sample from Homi Cape, Pohang city, South Korea, was a Gram-negative, aerobic, motile, non-spore-forming, rod- to ovoid-shaped bacterium. Colonies grown on marine agar were circular, convex and colourless to creamy white. Growth occurred between 10 and 40 °C (optimum 25–30 °C) and at pH 5.0–9.0 (optimum pH 6.0–8.0). The strain could grow in up to 15 % NaCl (optimum 3–5 % NaCl). According to 16S rRNA gene sequence analysis, the strain was a member of the genus Paracoccus in the Alphaproteobacteria. Sequence similarities to type strains of the genus Paracoccus were between 94.6 and 98.3 %, showing the highest sequence similarity to Paracoccus zeaxanthinifaciens ATCC 21588T. The DNA–DNA relatedness value of strain DD-R11T and P. zeaxanthinifaciens ATCC 21588T was 27 %. Strain DD-R11T was characterized by having ubiquinone 10 as the major respiratory quinone and C18 : 1{omega}7c as the predominant fatty acid. The DNA G+C content was 63.0 mol%. On the basis of its phenotypic and genotypic characteristics, it is suggested that DD-R11T represents a novel species of the genus Paracoccus, for which the name Paracoccus homiensis sp. nov. is proposed, with DD-R11T (=KACC 11518T=DSM 17862T) as the type strain.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DD-R11T is DQ342239.


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The genus Paracoccus was proposed by Davis et al. (1969)Go. At the time of writing, the genus contains 19 recognized species with validly published names. Phylogenetic analyses based on 16S rRNA gene sequences have shown that the genus Paracoccus falls within the {alpha}-3 subgroup of the Proteobacteria. Species of this genus are characterized as Gram-negative and catalase- and oxidase-positive, with a high metabolic versatility and a large amount of C18 : 1{omega}7c and having ubiquinone 10 as the major isoprenoid quinone.

In this study, a moderately halophilic strain, designated DD-R11T, was characterized on the basis of a polyphasic approach. The strain was isolated from a sea-sand sample collected in Pohang city, Korea. The sample was suspended in a solution of sea salts (Sigma), spread on marine agar 2216 (MA; Difco) and incubated at 28 °C. The isolate was routinely cultured on MA and maintained as a glycerol suspension (15 %, v/v) at –80 °C.

The isolate was cultivated on MA at 28 °C for the investigation of morphological and physiological characteristics. For observation of cell morphology by transmission electron microscopy, cells were grown for 48 h on MA, negatively stained with 0.5 % (w/v) uranyl acetate and examined with a LEO model 912AB electron microscope. Tolerance of pH and temperature was recorded following growth in marine broth at 28 °C for 21 days. The requirement for NaCl for growth was tested using nutrient broth supplemented with 0, 1, 3, 5, 7, 10, 15, 20 and 25 % (w/v) NaCl. Phenotypic characteristics such as Gram staining, catalase and oxidase production and hydrolysis of alginic acid, carboxymethylcellulose, casein, gelatin, pectin, tyrosine and starch were performed using the methods of Smibert & Krieg (1994)Go. Growth under anaerobic conditions was tested in GasPak (BBL) jars at 28 °C for 21 days. The commercially available API 20NE and API ZYM kits (bioMérieux) were used to determine biochemical properties, utilization of carbohydrates and enzymic activities according to the manufacturer's instructions. Utilization of organic substrates was tested using Biolog GN2 microplates. Colonies suspended in half-strength sea salts (Sigma) solution were used as the inoculum.

The 16S rRNA gene was amplified by PCR using two universal primers as described by Kwon et al. (2003)Go. The sequences of DD-R11T and related species were aligned using CLUSTAL X (Thompson et al., 1997Go). The evolutionary distances of 1394 aligned positions were computed using the Kimura two-parameter model (Kimura, 1980Go) and a phylogenetic tree was generated with MEGA version 2.1 (Kumar et al., 2001Go) using the neighbour-joining method (Saitou & Nei, 1987Go) and evaluated by bootstrap analyses (Felsenstein, 1985Go) based on 1000 resamplings.

DNA–DNA hybridization was carried out as described by Seldin & Dubnau (1985)Go. Probe labelling was conducted using the non-radioactive DIG-High Prime system (Roche); hybridized DNA was visualized using the DIG Luminescent Detection kit (Roche). DNA–DNA relatedness was quantified using a densitometer (Bio-Rad). Isoprenoid quinone analysis was performed as described by Groth et al. (1996)Go. The whole-cell fatty acid methyl ester profile was determined using the MIDI Sherlock Microbial Identification system (Microbial ID) with cells of DD-R11T grown at 28 °C for 24 h in MA. The DNA G+C content was determined according to the method of Mesbah et al. (1989)Go using a reversed-phase HPLC system with a C18 column.

Cells of strain DD-R11T were Gram-negative, non-spore-forming, aerobic and catalase- and oxidase-positive. Cells were rod- to ovoid-shaped (0.7–2.5x0.5–0.7 µm) and motile with one or more flagella (Fig. 1Go). They were able to grow on MA. However, they grew weakly on nutrient agar (Difco) and did not grow on trypticase soy agar (Difco) or MacConkey agar (Difco). The strain grew optimally on nutrient broth containing 3–5 % NaCl, grew weakly without NaCl and did not grow in the presence of more than 15 % (w/v) NaCl. After 48 h on MA, cells formed circular, convex, colourless to creamy white colonies. A phenotypic comparison of DD-R11T and closely related species of the genus Paracoccus is shown in Table 1Go.


Figure 1
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  		Fig. 1. Transmission electron micrograph of strain DD-R11T. Bar, 500 nm.

 

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  		Table 1. Phenotypic comparison of strain DD-R11T with closely related species within the genus Paracoccus

Taxa: 1, DD-R11T; 2, P. zeaxanthinifaciens (three strains); 3, P. carotinifaciens IFO 16121T; 4, P. haeundaensis KCCM 10460T; 5, P. marcusii DSM 11574T; 6, P. seriniphilus DSM 14827T. Data from Berry et al. (2003)Go, Pukall et al. (2003)Go, Lee et al. (2004)Go and this study. +, Positive; –, negative; W, weak; ND, not determined.

 
The major fatty acid for strain DD-R11T was C18 : 1{omega}7c (78.9 %). Small amounts of other fatty acids were detected as follows: 11-methyl C18 : 1{omega}7c (5.8 %), C18 : 0 (4.5 %), C10 : 0 3-OH (2.6 %), unidentified ECL 11.799 (2.4 %), C12 : 0 ALDE (1.8 %) and C18 : 0 3-OH (1.1 %). This fatty acid profile was similar to those of members of the genus Paracoccus (Kelly et al., 2000Go) (Table 2Go).


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  		Table 2. Fatty acid composition of DD-R11T and its nearest phylogenetic neighbours within the genus Paracoccus

Taxa: 1, DD-R11T; 2, P. zeaxanthinifaciens (mean values from three strains); 3, P. carotinifaciens IFO 16121T; 4, P. haeundaensis KCCM 10460T; 5, P. marcusii DSM 11574T; 6, P. seriniphilus DSM 14827T. Data from Berry et al. (2003)Go, Pukall et al. (2003)Go, Lee et al. (2004)Go and this study. Values are percentages of total fatty acids; –, not detected or <1 %.

 
16S rRNA gene sequencing showed that the sequence of strain DD-R11T exhibited 94.6–98.3 % sequence similarity to type strains of the genus Paracoccus. Strain DD-R11T showed the highest sequence similarity to Paracoccus zeaxanthinifaciens ATCC 21588T (98.3 %), followed by Paracoccus seriniphilus DSM 14827T (96.7 %). The phylogenetic tree showing the position of strain DD-R11T relative to members of the genus Paracoccus is shown in Fig. 2Go. Strain DD-R11T and P. zeaxanthinifaciens ATCC 21588T formed a compact cluster (100 % bootstrap value), which was loosely related to another cluster including Paracoccus seriniphilus DSM 14827T, Paracoccus carotinifaciens IFO 16121T, Paracoccus marcusii DSM 11574T and Paracoccus haeundaensis KCCM 10460T.


Figure 2
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  		Fig. 2. Neighbour-joining tree showing the relationship of strain DD-R11T to other members of the genus Paracoccus based on 16S rRNA gene sequences. Only bootstrap percentages above 50 % are shown (1000 replications). The sequence of Rhodobacter capsulatus ATCC 11166T was used as the outgroup. Bar, 0.5 % sequence divergence.

 
According to DNA–DNA hybridization, strain DD-R11T showed a DNA–DNA relatedness value of 27 % to P. zeaxanthinifaciens ATCC 21588T. The levels obtained for 16S rRNA gene similarity (<97 %) and DNA–DNA hybridization (<70 %) support the genomic distinction of strain DD-R11T from other species in the genus Paracoccus (Stackebrandt & Goebel, 1994Go; Wayne et al., 1987Go). On the basis of the phylogenetic, chemotaxonomic and phenotypic data, strain DD-R11T should be classified as the type strain of a novel species of the genus Paracoccus, for which we propose the name Paracoccus homiensis sp. nov.

Description of Paracoccus homiensis sp. nov.
Paracoccus homiensis (ho.mi.en'sis. N.L. masc. adj. homiensis pertaining to Homi Cape, South Korea, where the type strain was isolated).

Cells are Gram-negative, aerobic, motile, non-spore-forming and rod- to ovoid-shaped (0.7–2.5x0.5–0.7 µm). Colonies on MA are circular, convex and colourless to creamy white. Growth occurs between 10 and 40 °C (optimum 25–30 °C) and at pH 5.0–9.0 (optimum pH 6.0–8.0). Grows optimally at 3–5 % NaCl and weakly at 15 % (w/v) NaCl. Positive for catalase, oxidase and hydrolysis of aesculin, gelatin, Tween 80 and tyrosine. Negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, hydrolysis of alginic acid, casein, carboxymethylcellulose, pectin, starch and urea. Enzymic activity is observed for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, {alpha}-galactosidase, beta-galactosidase and {alpha}-glucosidase, but no activity is observed for lipase (C14), valine arylamidase, trypsin, {alpha}-chymotrypsin, beta-glucuronidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, {alpha}-mannosidase and {alpha}-fucosidase. Utilizes Tween 80, D-arabitol, D-cellobiose, iso-erythritol, D-fructose, D-galactose, {alpha}-D-glucose, myo-inositol, {alpha}-D-lactose, D-mannitol, D-mannose, D-melibiose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, pyruvic acid methyl ester, succinic acid monomethyl ester, acetic acid, cis-aconitic acid, citric acid, formic acid, D-gluconic acid, D-glucuronic acid, beta-hydroxybutyric acid, p-hydroxyphenylacetic acid, DL-lactic acid, propionic acid, D-saccharic acid, succinic acid, D-alanine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-serine, phenylethylamine and glycerol. Does not utilize {alpha}-cyclodextrin, dextrin, glycogen, Tween 40, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, L-arabinose, L-fucose, gentiobiose, lactulose, maltose, methyl beta-D-glucoside, D-psicose, xylitol, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, {alpha}-hydroxybutyric acid, {gamma}-hydroxybutyric acid, itaconic acid, {alpha}-ketobutyric acid, {alpha}-ketoglutaric acid, {alpha}-ketovaleric acid, malonic acid, quinic acid, sebacic acid, bromosuccinic acid, succinamic acid, glucuronamide, L-alaninamide, L-alanyl glycine, glycyl L-aspartic acid, glycyl L-glutamic acid, L-histidine, hydroxy-L-proline, L-pyroglutamic acid, D-serine, L-threonine, DL-carnitine, {gamma}-aminobutyric acid, urocanic acid, inosine, uridine, thymidine, putrescine, 2-aminoethanol, 2,3-butanediol, DL-{alpha}-glycerol phosphate, {alpha}-D-glucose 1-phosphate and D-glucose 6-phosphate. Ubiquinone Q-10 is the major isoprenoid quinone. The major fatty acid is C18 : 1{omega}7c. The G+C content is 63.0 mol%.

The type strain, DD-R11T (=KACC 11518T=DSM 17862T), was isolated from a sea-sand sample collected from Homi Cape, Pohang city, South Korea.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant (203068-03-2-SB010) from the Agricultural R&D Promotion Center, South Korea.


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Davis, D. H., Doudoroff, M., Stanier, R. Y. & Mandel, M. (1969). Proposal to reject the genus Hydrogenomonas: taxonomic implications. Int J Syst Bacteriol 19, 375–390.[Abstract/Free Full Text]

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Kelly, D. P., Rainey, F. A. & Wood, A. P. (2000). The genus Paracoccus. In The Prokaryotes: an Evolving Electronic Resource for the Microbiological Community, 3rd edn. Edited by M. Dworkin, N. Falkow, H. Rosenberg, K.-H. Schleifer & E. Stackebrandt. New York: Springer-Verlag.

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