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1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
2 National University of Uzbekistan, VUZ-gorodok, Tashkent 700-174, Uzbekistan
Correspondence
Sung-Taik Lee
e_stlee{at}kaist.ac.kr
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-Proteobacteria, being related to Paracoccus solventivorans (97·5 %), Paracoccus alkenifer (96·9 %) and Paracoccus kocurii (96·4 %). The phylogenetic distance from Paracoccus species with validly published names was always less than 96 %. Physiological and chemotaxonomic data (major ubiquinone, Q-10; major fatty acids, C18 : 1 and C18 : 0) supported the affiliation of strain Ch05T to the genus Paracoccus. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Ch05T from the 17 Paracoccus species with validly published names. Ch05T therefore represents a novel species, for which the name Paracoccus koreensis sp. nov. is proposed. The type strain is Ch05T (=KCTC 12238T=IAM 15216T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Ch05T is AB187584.
Present address: School of Civil, Urban and Geosystem Engineering, Seoul National University, Shillim-dong, Gwanak-gu, Seoul, Republic of Korea. 
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). One of these isolates (strain Ch05T) was found to be a member of the genus Paracoccus in the -Proteobacteria lineage and was the subject of a taxonomic investigation.
The genus Paracoccus was first described by Davis et al. (1969); the genus currently comprises 17 species with validly published names. The members of the genus Paracoccus consist of Gram-negative cocci or short rods that show substantial metabolic versatility. Representatives are able to grow aerobically on a wide range of organic compounds. A number of species can also grow anaerobically, using nitrate as electron acceptor, and some representatives are able to use hydrogen as electron donor for chemoautotrophic growth. Phylogenetically, the genus belongs to the -3 subgroup of the Proteobacteria. Recently, Paracoccus seriniphilus (Pukall et al., 2003), Paracoccus yeei (Daneshvar et al., 2003) and Paracoccus zeaxanthinifaciens (Berry et al., 2003) were introduced.
Strain Ch05T was isolated from granules involved in the treatment of wastewater from a beer-brewing factory in Chung-Ju, Republic of Korea. Anaerobic granules are bacterial aggregates that result from the flocculation of sludge in an upflow anaerobic sludge blanket (UASB) reactor (de Zeeuw & Lettinga, 1980; Lettinga, 1995). They are composed of micro-organisms, inorganic nuclei and extracellular polymers (Fukuzaki et al., 1991; Shen et al., 1993). Great attention has been paid to the internal structure and catalytic activities of such granules (MacLeod et al., 1990; Schmidt & Ahring, 1996). In our laboratory, the relationship between the structure and the resistance to toxic chemicals in anaerobic granules from a brewery-wastewater-treatment UASB reactor was studied (Bae & Lee, 1999; Bae et al., 2000). In a series of studies, we attempted to isolate micro-organisms from the anaerobic granules in order to investigate the community structure by using a culture system. Interestingly, the granules contained aerobic bacteria even though they had been kept under anaerobic conditions for 2 years. Strain Ch05T is one of the dominant bacterial isolates grown under aerobic conditions.
In the present study, we conducted a phylogenetic analysis on the basis of 16S rRNA gene sequences, DNA–DNA relatedness and some important phenotypic characteristics to determine the precise taxonomic position of this strain. On the basis of the results obtained in this study, we propose that strain Ch05T should be placed in the genus Paracoccus as the type strain of a novel species.
For the isolation of aerobic bacteria, brownish black granules (around 2 mm in diameter) from a brewery-wastewater-treating UASB reactor, which had been operated anaerobically for 2 years, were homogenized by using an Ace homogenizer (Nihonseiki). The suspension was spread on R2A agar plates (Scharlau) after being serially diluted with 50 mM phosphate buffer (pH 7·0). The plates were incubated at 30 °C for 2 weeks. Single colonies on the plates were purified by transferring them onto new plates and incubating them once again under the same conditions. The purified colonies were tentatively identified by means of partial sequences of 16S rRNA. Ch05T was one of the dominant isolates appearing on the plates under aerobic conditions.
A Gram-reaction was performed by using the non-staining method as described by Buck (1982). Cell morphology was observed under a Nikon E600 light microscope at x1000, using cells grown for 2 days at 30 °C on R2A agar. Catalase and oxidase tests were performed by using the procedures outlined by Cappuccino & Sherman (2002). The utilization of various substrates as sole carbon sources, and some physiological characteristics, were determined with API 32GN and API 20NE galleries according to the instructions of the manufacturer (bioMérieux). Nitrate and nitrite reduction were later confirmed by inoculating cells, in each case, into three serum bottles (25 ml) containing 13 ml R2A medium, while nitrate and nitrite were added as KNO3 and NaNO2 at concentrations of 10 mM. The reduction of nitrate and nitrite was monitored by ion chromatograph (model 790 personal IC; Metrohm) equipped with a conductivity detector and anion exchange column (Metrosep Anion Supp 4; Metrohm). The physiological characteristics of strain Ch05T and related type strains are summarized in Table 1.
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. The 16S rRNA gene sequences of related taxa were obtained from GenBank. The multiple alignments were performed using the CLUSTAL X program (Thompson et al., 1997). Gaps were edited in the BioEdit program (Hall, 1999). Evolutionary distances were calculated using Kimura's two-parameter model (Kimura, 1983). A phylogenetic tree was constructed by using the neighbour-joining methods (Saitou & Nei, 1987) in the MEGA2 program (Kumar et al., 2001), with bootstrap values based on 1000 replications (Felsenstein, 1985) (Fig. 1). The 16S rRNA gene sequence of strain Ch05T was a continuous stretch of 1386 bp. Sequence-similarity calculations after neighbour-joining analysis indicated that the closest relatives of strain Ch05T were Paracoccus solventivorans (97·5 %), Paracoccus alkenifer (96·9 %) and Paracoccus kocurii (96·4 %), and the phylogenetic distance from other Paracoccus species with validly published names was more than 4 %.
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, using reverse-phase HPLC. Quinones were extracted from cells grown on a nutrient broth (Scharlau) and then analysed as described by Komagata & Suzuki (1987), using reverse-phase HPLC. Cellular fatty acids were analysed in micro-organisms grown on trypticase soy agar (Scharlau) for 2 days. The cellular fatty acids were saponified, methylated and extracted according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids were analysed using gas chromatography (6890; Hewlett Packard) and identified by using the Microbial Identification software package (Sasser, 1990).
The G+C content of genomic DNA of strain Ch05T was 69 mol%, while those of Paracoccus species were in the range 63–71 mol% (Harker et al., 1998). The respiratory quinone system of Ch05T supports the affiliation of this strain to the -Proteobacteria, as the majority of Paracoccus species contain Q-10 as the major quinone. The fatty acid profile of strain Ch05T was composed mainly of C18 : 1 (79·6 %), C18 : 0 (13·4 %), C10 : 0 3-OH (2·4 %) and C16 : 0 (2·3 %). A comparison of the fatty acid profile of Ch05T with those of phylogenetically related strains is presented in Table 2. The fatty acid profile of strain Ch05T is relatively simple compared with the others.
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with photobiotin-labelled DNA probes and microdilution wells. The DNA–DNA relatedness values for strain Ch05T with respect to the type strains of the phylogenetically closest Paracoccus species were in the range 14–36 %, which is sufficiently low to allow the assignment of strain Ch05T to the genus Paracoccus as a novel species. On the basis of the data and observations described above, strain Ch05T should be assigned to the genus Paracoccus as the type strain of a novel species, for which the name Paracoccus koreensis sp. nov. is proposed.
Description of Paracoccus koreensis sp. nov.
Paracoccus koreensis (ko.re.en'sis. N.L. masc. adj. koreensis pertaining to Korea, the location of the granule samples from which the type strain was isolated).
Cells are Gram-negative, aerobic, non-motile, short rod- to coccus-shaped and 0·5–1·0 µm by 1·0–1·5 µm in size after 2 days culture on R2A agar. Colonies grown on R2A agar for 1–2 days are smooth, circular, non-glossy, creamy white and convex. Grows well at 15–37 °C and at pH 6–8, but does not grow at 4 or 45 °C. Catalase- and oxidase-positive; shows oxidative metabolism. Unable to grow anaerobically using nitrate as electron acceptor. Unable to reduce nitrate but able to reduce nitrite. Substrate utilization, enzyme production, acid production and other physiological characteristics are indicated in Table 1
. Q-10 is the predominant ubiquinone. C18 : 1 (79·6 %) and C18 : 0 (13·4 %) are the major cellular fatty acids. The G+C content of the genomic DNA is 69 mol% (as determined by HPLC).
The type strain, Ch05T (=KCTC 12238T=IAM 15216T), was isolated from granules used in the wastewater-treatment plant of a beer-brewing factory in Chung-Won, Republic of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bae, J.-W., Rhee, S.-K., Hyun, S.-H., Kim, I. S. & Lee, S.-T. (2000). Layered structure of granules in upflow anaerobic sludge blanket reactor gives microbial populations resistance to metal ions. Biotechnol Lett 22, 1935–1940.[CrossRef]
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Buck, J. D. (1982). Nonstaining (KOH) method for determination of Gram reactions of marine bacteria. Appl Environ Microbiol 44, 992–993.
Cappuccino, J. G. & Sherman, N. (2002). Microbiology: a Laboratory Manual, 6th edn. San Francisco: Pearson Education.
Daneshvar, M. I., Hollis, D. G., Weyant, R. S. & 11 other authors (2003). Paracoccus yeeii sp. nov. (formerly CDC group EO-2), a novel bacterial species associated with human infection. J Clin Microbiol 41, 1289–1294.
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-glucosidase, 