Paracoccus aestuarii sp. nov., isolated from tidal flat sediment

A Gram-negative micro-organism, designated strain B7^T, was isolated from tidal flat sediment and subjected to a polyphasic taxonomic study involving morphological, physiological, biochemical and 16S rRNA gene sequence analyses. A phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain B7^T belonged to the genus Paracoccus and was closely related phylogenetically to Paracoccus marcusii MH1^T (97.5 % sequence similarity), Paracoccus marinus KKL-A5^T (97.5 %), Paracoccus haeundaensis BC74171^T (97.3 %), Paracoccus carotinifaciens E-396^T (97.3 %), Paracoccus homiensis DD-R11^T (97.2 %), Paracoccus seriniphilus MBT-A4^T (96.9 %) and other type strains of the genus Paracoccus (95.2–96.7 %). The G+C content of the genomic DNA and the major isoprenoid quinone of the type strain were 62.0 mol% and ubiquinone-10, respectively. The major fatty acid components were C_{18 : 1}ω7c (68.9 %) and C_{18 : 0} (18.1 %); this profile, with C_{18 : 1}ω7c as the predominant fatty acid, was characteristic of members of the genus Paracoccus. The 16S rRNA gene sequence analysis, DNA–DNA hybridization studies and physiological and biochemical tests identified genotypic and phenotypic differences between strain B7^T and recognized Paracoccus species. On the basis of these data, therefore, strain B7^T represents a novel species of the genus Paracoccus, for which the name Paracoccus aestuarii sp. nov. is proposed. The type strain is B7^T (=KCTC 22049^T=DSM 19484^T=JCM 15119^T).

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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain B7^T is EF660757.

The genus Paracoccus belongs to the Alphaproteobacteria and was first proposed by Davis et al. (1969) as comprising a Gram-negative, catalase-positive, oxidase-positive bacterium, Paracoccus denitrificans. Members of this genus contain C_{18 : 1}ω7c as a major component of the cellular fatty acids and are metabolically versatile. At the time of writing, since this genus was proposed in 1969, the names of 24 species belonging to the genus Paracoccus have been validly published, having been isolated from various environments including soil, sediment and activated sludge. The purpose of this paper was to establish the taxonomic position of strain B7^T, which was isolated from tidal flat sediment, on the basis of data from phenotypic, genetic and chemotaxonomic analyses.

Strain B7^T was isolated from tidal flat sediment in Yeosu (3 ° 47' 26'' N 12 ° 34' 01'' E), South Korea. This novel strain was isolated on TSBA (tryptic soy broth solidified with 20.0 g agar l^–1; Difco) and a pure culture was obtained by using repeated restreaking. Cell biomass for analysis of cellular composition and for DNA extraction was taken from TSBA plates that had been incubated at 30 °C for 2 days.

A DNA-extraction kit (G-spin, iNtRON Biotechnology) was used to obtain chromosomal DNA. Two universal bacterial primers: forward primer 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492r (5'-TACGGYTACCTTGTTACGACTT-3') (Lane, 1991) were used to PCR-amplify the 16S rRNA gene from the chromosomal DNA. After purification with a PCR purification kit (Solgent), the PCR product was sequenced as described previously (Roh et al., 2008). SeqMan software (DNASTAR) was used to assemble full-length 16S rRNA gene sequences. The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (; Chun et al., 2007). 16S rRNA gene sequences from the novel isolate and related taxa (NCBI database) were aligned using the multiple sequence alignment program CLUSTAL_X (version 1.8) (Thompson et al., 1997). Phylogenetic relationships between representative Paracoccus species were determined using the MEGA3 software program (Kumar et al., 2004). Dendrograms were elaborated by means of distance matrices (Kimura, 1980), using the neighbour-joining method (Saitou & Nei, 1987). To evaluate the stability of the trees, a bootstrap analysis was performed using a consensus tree that was based on 1000 randomly generated trees. DNA–DNA hybridization was performed using the fluorometric method of Ezaki et al. (1989). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain B7^T falls within the cluster of Paracoccus species (Fig. 1) and is closely related phylogenetically to Paracoccus marcusii MH1^T (97.5 % sequence similarity), Paracoccus marinus KKL-A5^T (97.5 %), Paracoccus haeundaensis BC74171^T (97.3 %), Paracoccus carotinifaciens E-396^T (97.3 %), Paracoccus homiensis DD-R11^T (97.2 %), Paracoccus seriniphilus MBT-A4^T (96.9 %) and other type strains of the genus Paracoccus (95.2–96.7 %). A DNA–DNA hybridization study revealed the following mean levels of DNA–DNA relatedness between strain B7^T and the type strains of its closest relatives in the phylogenetic tree: P. marcusii (9.4 %), P. homiensis (13.9 %) and P. seriniphilus (11.3 %). P. haeundaensis BC74171^T and P. carotinifaciens E-396^T are patent strains that are not publicly available, so a DNA–DNA relatedness study could not be conducted with these two strains. Species definitions can be established with DNA–DNA reassociation values below 70 % (Wayne et al., 1987), and 16S rRNA gene sequence similarity values that are less than 98.7 % between two strain pairs have DNA–DNA reassociation values of less than 70 % (Stackebrandt & Ebers, 2006); therefore, strain B7^T can be considered to represent a distinct genospecies.

(55K): Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain B7T with respect to species in the genus Paracoccus. Numbers at nodes indicate bootstrap percentages (based on 1000 replications), where greater than 50 %. Bar, 0.005 accumulated changes per nucleotide.

The DNA G+C content was determined using HPLC as described by Mesbah & Whitman (1989), with Escherichia coli B (Sigma-Aldrich) as the calibration reference. The G+C content of the genomic DNA of the type strain was 62.0 mol%. The lowest genomic DNA G+C content among species belonging to the genus Paracoccus is 61.3 mol% (for the type strain of Paracoccus sulfuroxidans; Liu et al., 2006) and the highest value is 71.0 mol% (for the type strain of Paracoccus kocurii; Ohara et al., 1990). The G+C content of the genomic DNA of strain B7^T is therefore within the range for recognized Paracoccus species.

The quantitative analysis of the cellular fatty acid composition was performed according to the instructions for the Sherlock Microbial Identification System (MIDI). Gas chromatography (6890; Hewlett Packard) together with the Microbial Identification software package (Sasser, 1990) were used to identify the fatty acids. The cellular fatty acids of strain B7^T were C_{18 : 1}ω7c (68.9 %), C_{18 : 0} (18.1 %), C_{10 : 0} 3-OH (2.5 %), C_{17 : 0} (2.5 %), C_{16 : 0} (1.3 %), summed feature 2 (comprising C_{14 : 0} 3-OH and/or iso-C_{16 : 1} I; 2.4 %) and some others (<1 %). This profile, with C_{18 : 1}ω7c as the major fatty acid, is characteristic of members of the genus Paracoccus. However, the C_{18 : 1}ω7c (68.9 %) and C_{18 : 0} (18.1 %) contents of strain B7^T differed from the ranges (78.9–84.3 % and 2.5–7.8 %, respectively) found among the Paracoccus species (P. marcusii, P. haeundaensis, P. carotinifaciens, P. seriniphilus, P. homiensis and P. zeaxanthinifaciens; Kim et al., 2006) that clustered with the novel strain in the phylogenetic tree. The differences in fatty acid composition may be the result of differences in experimental conditions.

Quinones were characterized as described by Collins (1985) and Wu et al. (1989). Paracoccus species are characterized as possessing ubiquinone-10 (Q-10). Strain B7^T also possessed Q-10 as the sole respiratory quinone. This result, in combination with the 16S rRNA gene sequence data, the G+C content and the major fatty acid components, confirmed that the novel strain belongs to the genus Paracoccus.

Polar lipids were extracted and then examined by means of two-dimensional TLC followed by spray reagents (Collins et al., 1980). The polar lipid analysis indicated that strain B7^T contained phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol.

To determine the optimum growth temperature for strain B7^T, cells were grown on TSBA plates at 4–45 °C. The optimum NaCl concentration and pH were determined in TSB medium supplemented with NaCl (0–30 %, w/v) and with HCl or NaOH (pH 4.0–13.0). Cell morphology was examined under light microscopy (ECLIPSE 80i; Nikon). Motility was observed using the wet-mount method (Murray et al., 1994) and Gram staining was performed with the non-staining method described by Buck (1982). Anaerobic growth was tested on TSBA plates using the BBL GasPak Pouch System (Becton Dickinson). Enzyme activities and substrate utilization from sole carbon sources were determined using API ZYM, API 20NE and API 50 CH test strips (bioMérieux). Catalase activity was investigated by assessing bubble production in a 3 % (v/v) hydrogen peroxide solution. A detailed species description is presented below and Table 1 shows a comparison between the characteristics of strain B7^T and those of six closely related strains (selected on the basis of 16S rRNA gene sequence similarity and phylogenetic tree topology). Our detailed investigation has revealed genotypic and phenotypic differences between strain B7^T and recognized Paracoccus species.

Table 1. Characteristics that serve to differentiate strain B7T from closely related species Strains: 1, B7T (P. aestuarii sp. nov.); 2, P. marcusii MH1T (patent strain; data from Harker et al., 1998); 3, P. haeundaensis BC74171T (patent strain; Lee et al., 2004); 4, P. carotinifaciens E-396T (patent strain; Tsubokura et al., 1999); 5. P. seriniphilus MBT-A4T (Pukall et al., 2003); 6. P. homiensis DD-R11T (Kim et al., 2006); 7. P. zeaxanthinifaciens R-1512T (Berry et al., 2003). +, Positive; –, negative; W, weakly positive; NR, not reported.

Thus, on the basis of genetic, chemotaxonomic and phenotypic comparisons with previously described taxa, strain B7^T represents a novel species of the genus Paracoccus, for which the name Paracoccus aestuarii sp. nov. is proposed.

Description of Paracoccus aestuarii sp. nov.
Paracoccus aestuarii (a.es.tu.a.ri'i. L. gen. n. aestuarii of a tidal flat).

Cells are aerobic, non-motile, rod-shaped (0.8–1.0x1.5–2.0 µm), Gram-negative, catalase-positive and oxidase-positive. Colonies are orange-coloured, circular and approximately 1.0–2.5 mm in diameter after growth for 1 day on TSBA at 30 °C. No growth occurs at NaCl concentrations greater than 5 %. The temperature range for growth is 15–37 °C. The pH range for growth is 7.5–9.5 (optimum, pH 9.0). Nitrate is not reduced to nitrite or nitrogen, indole is not produced and glucose fermentation does not occur. Negative for arginine dihydrolase and urease. Aesculin and PNPG (p-nitrophenyl-β-D-galactopyranoside) hydrolysis occurs, but not gelatin hydrolysis. D-Glucose, D-mannose, D-mannitol, N-acetylglucosamine, maltose, D-malate, trisodium citrate, D-ribose, D-xylose, D-adonitol, inositol, arbutin, aesculin, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate are assimilated, but potassium gluconate, capric acid, adipic acid, phenylacetic acid, glycerol, erythritol, D-arabinose, L-arabinose, L-xylose, methyl β-D-xyloside, D-galactose, D-fructose, L-sorbose, L-rhamnose, dulcitol, D-sorbitol, methyl α-D-mannoside, methyl α-D-glucoside, amygdalin, salicin, cellobiose, D-lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, turanose, D-lyxose, D-tagatose, D-fucose and L-fucose are not assimilated. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. Negative for lipase (C14), cystine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Predominant fatty acids are C_{18 : 1}ω7c, C_{18 : 0}, C_{10 : 0} 3-OH, C_{17 : 0}, C_{16 : 0} and summed feature 2 (comprising C_{14 : 0} 3-OH and/or iso-C_{16 : 1} I). The polar lipid fraction consists of phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol. The major isoprenoid quinone is Q-10. The G+C content of genomic DNA of the type strain is 62.0 mol%.

The type strain, B7^T (=KCTC 22049^T=DSM 19484^T=JCM 15119^T), was isolated from tidal flat sediment in Yeosu (3 ° 47' 26'' N 12 ° 34' 01'' E), South Korea.