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Int J Syst Evol Microbiol 58 (2008), 139-143; DOI  10.1099/ijs.0.65346-0
© 2008 International Union of Microbiological Societies

Actibacter sediminis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment

Ju-Hyoung Kim1, Ki-Youn Kim1, Young-Tae Hahm1, Bong-Soo Kim2, Jongsik Chun2 and Chang-Jun Cha1

1 Department of Biotechnology and BET Institute, Chung-Ang University, Anseong 456-756, Republic of Korea
2 School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea

Correspondence
Chang-Jun Cha
cjcha{at}cau.ac.kr


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A yellow-pigmented, Gram-negative, aerobic bacterial strain comprising rod-shaped cells devoid of flagellar and gliding motility, designated strain JC2129T, was isolated from tidal flat sediment of Dongmak on Ganghwa Island, South Korea. Results from a 16S rRNA gene sequence analysis indicated that the isolate belonged to the family Flavobacteriaceae; the highest level of nucleotide sequence similarity (91.9 %) occurred with Polaribacter dokdonensis DSW-5T. The predominant cellular fatty acids were iso-C15 : 0 (19.8 %), iso-C15 : 1 G (14.0 %), iso-C17 : 0 3-OH (13.7 %) and iso-C13 : 0 (6.4 %). Flexirubin-type pigments were absent. The major isoprenoid quinone was MK-6. The DNA G+C content was 43–45 mol%. Data from a polyphasic taxonomy study suggested that the isolate represents a novel genus and species in the family Flavobacteriaceae, for which the name Actibacter sediminis gen. nov., sp. nov. is proposed. The type strain of Actibacter sediminis is JC2129T (=KCTC 12704T =JCM 14002T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of the strain JC2129T is EF670651.

A transmission electron micrograph of a negatively stained cell of strain JC2129T is shown as a supplementary figure available with the online version of this paper.


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Tidal flats in Korea have been known to harbour a highly diverse prokaryotic community, as shown by both culture-dependent and culture-independent surveys (Kim et al., 2004Go, 2005Go; Yi & Chun, 2006Go). In this study, we report the description of a novel member of this bacterial community, a Flavobacterium-like bacterium that showed low levels of 16S rRNA gene sequence similarity with respect to members of the family Flavobacteriaceae with validly published names.

A bacterial strain, designated JC2129T, was isolated from a sample of tidal flat sediment from Ganghwa Island, South Korea (3 ° 36' 22.3'' N 12 ° 22' 59.4'' E), using the standard dilution plating method on marine agar 2216 (MA; Conda). The isolate was routinely cultured on MA and maintained at –80 °C as a suspension in marine broth (MB; Conda) supplemented with 20 % (v/v) glycerol.

The 16S rRNA gene was amplified enzymically from a single colony by means of a PCR using AccuPower PCR Premix (Bioneer) and primers 27F and 1492R (Lane, 1991Go). The PCR product was purified using an AccuPrep PCR purification kit (Bioneer) and the sequencing of the 16S rRNA gene was performed with an Applied Biosystems ABI3730XL automatic sequencer at Macrogen Corp. (Seoul, South Korea). The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (http://www.eztaxon.org/; Chun et al., 2007Go). The initial similarity analyses indicated that our isolate belonged to the family Flavobacteriaceae. The almost-complete 16S rRNA gene sequence of strain JC2129T (1374 bp) was aligned manually against those of representatives of the family Flavobacteriaceae using the bacterial 16S rRNA secondary structure model and the jPHYDIT program (Jeon et al., 2005Go). The phylogenetic tree was inferred by using the neighbour-joining method (Saitou & Nei, 1987Go) on the basis of distance matrix data. Evolutionary distance matrices for the neighbour-joining method were generated according to the model of Jukes & Cantor (1969)Go. The resultant neighbour-joining tree topology was evaluated by means of bootstrap analyses (Felsenstein, 1985Go) based on 1000 resamplings. Phylogenetic analyses were carried out using the MEGA3 (Kumar et al., 2004Go) and PHYLIP (Felsenstein, 2005Go) programs.

On the basis of the 16S rRNA gene sequence analyses, strain JC2129T showed low levels of similarity with respect to known species of the family Flavobacteriaceae. The highest level of sequence similarity was found with respect to Polaribacter dokdonensis DSW-5T (91.9 %), followed by Tenacibaculum litoreum CL-TF13T (91.9 %) and Lutibacter litoralis CL-TF09T (91.6 %). Phylogenetic treeing (Fig. 1Go) also confirmed that the tidal flat isolate JC2129T is a member of the family Flavobacteriaceae but distantly associated with the aforementioned genera. The tree suggested that strain JC2129T formed a monophyletic clade with the genera Lutibacter, Polaribacter and Tenacibaculum, with 99 % bootstrap support.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree, based on almost-complete 16S rRNA gene sequences, showing the relationships between strain JC2129T and representative members of the family Flavobacteriaceae. Bootstrap percentages greater than 50 % (based on neighbour-joining analyses of 1000 resampled datasets) are shown at nodes. Flexibacter flexilis ATCC 23079T (M62794) (not shown) was used as an outgroup. Bar, 0.05 nucleotide substitutions per position.

 
The cellular morphology was examined using transmission electron microscopy, after growth of strain JC2129T on MA at 30 °C for 2 days. Gliding motility was investigated by examining the edge of a hanging drop of a fresh MB culture, as recommended by Bernardet et al. (2002)Go. Anaerobic growth was evaluated on MA in an anaerobic-chamber system (Coy Laboratory Products). The pH range (3–10, using increments of 1 pH unit) for growth was determined using MB; the final pH was adjusted with NaOH and HCl solutions after autoclaving. The requirements for NaCl (0–10 %, using increments of 1 %, w/v) and sea salts (0, 1, 3, 5, 7, 10, 15 and 20 %, w/v; Sigma) were tested in R2A medium (Conda). The temperature range for growth was determined on MA at 5–50 °C (using increments of 5 °C). Hydrolysis of casein, starch and Tween 80 was tested using MA, as described by Smibert & Krieg (1994)Go. Chitin hydrolysis was tested on chitin agar as described by Hsu & Lockwood (1975)Go. Cellulase activity was tested by observing the disintegration of filter paper in an MB culture (Smibert & Krieg, 1994Go; Bernardet et al., 2002Go). DNase test agar (Difco) supplemented with 2.5 % (w/v) NaCl was used for a DNase assay. H2S production was detected in triple-sugar–iron agar supplemented with 2.5 % (w/v) NaCl. Arginine dihydrolase, β-galactosidase, nitrate reduction, urease, acid production from glucose and indole-production tests were performed using an API 20NE kit (bioMérieux) according to the manufacturer's instructions; other enzymic activities were determined using an API ZYM kit (bioMérieux). Kits were inoculated with a heavy bacterial suspension in half-strength artificial seawater (24 g NaCl, 5.1 g MgCl2, 4 g Na2SO4, 1.1 g CaCl2, 0.7 g KCl, 0.2 g NaHCO3, 0.1 g KBr, 0.027 g H3BO3, 0.024 g SrCl2 and 0.003 g NaF per litre distilled water; Lyman & Fleming, 1940Go) and AUX media (bioMérieux) supplemented with 2.5 % (w/v) NaCl. Carbon-source utilization was tested by incubating strain JC2129T at 37 °C for 2 weeks on basal agar medium supplemented with yeast extract (0.64 g KCl, 23.6 g NaCl, 5.94 g MgSO4 . 7H2O, 4.53 g MgCl2 . 6H2O, 1.3 g CaCl2 . 2H2O, 0.2 g NH4Cl, 0.2 g NaNO3, 15 g Bacto agar and 0.05 g yeast extract per litre distilled water; Choi & Cho, 2006Go) containing 0.2 % carbon source.

A transmission electron micrograph of strain JC2129T is available as Supplementary Fig. S1 in IJSEM Online. Its biochemical and physiological properties are presented in Table 1Go and in the genus and species descriptions.


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  		Table 1. Differential characteristics of strain JC2129T and related organisms in the family Flavobacteriaceae

Taxa: 1, strain JC2129T; 2, Polaribacter (data for six species); 3, Tenacibaculum (nine species); 4, Lutibacter litoralis. Data are from Gosink et al. (1998)Go, Suzuki et al. (2001)Go, Frette et al. (2004)Go, Yoon et al. (2005)Go, Choi et al. (2006)Go, Choi & Cho (2006)Go and this study. +, Positive; –, negative; V, variable among species; NA, not available.

 
Chemotaxonomic characteristics were determined from cells grown at 30 °C for 2–3 days on MA or in MB. Analysis of the fatty acid methyl esters was carried out by GLC according to the instructions of the Microbial Identification system (MIDI). Isoprenoid quinones were isolated by using the method of Minnikin et al. (1984)Go and were analysed by HPLC (Varian) as described by Collins (1985)Go. The DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Mesbah et al. (1989)Go, using a reversed-phase column (Supelcosil LC-18 S; Supelco). Experiments were performed in triplicate. Flexirubin-type pigments were sought by using the KOH test as described by Bernardet et al. (2002)Go.

The cellular fatty acid profiles of strain JC2129T and related members of the family Flavobacteriaceae are shown in Table 2Go. The major respiratory quinone was MK-6, but a rather large amount of MK-7 was also found (MK-7/MK-6 ratio, 1 : 2.8). Flexirubin-type pigments were not detected. The DNA G+C content of strain JC2129T was found to be 43–45 mol%.


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  		Table 2. Cellular fatty acid compositions of strain JC2129T and type strains of related organisms in the family Flavobacteriaceae

Strains: 1, JC2129T; 2, Polaribacter filamentus 215T (data from Gosink et al., 1998Go); 3, Tenacibaculum maritimum JCM 8137T (Yoon et al., 2005Go); 4, L. litoralis JCM 13034T (Choi & Cho, 2006Go). Strain JC2129T, T. maritimum JCM 8137T and L. litoralis JCM 13034T were grown on MA at 30 °C for 1–2 days, whereas P. filamentus 215T was grown on seawater–cytophaga medium at 5 °C for 3–4 weeks. Values are percentages of total fatty acids; components amounting to less than 1 % of the total fatty acids in all strains are not included. –, Not detected/not reported.

 
Strain JC2129T exhibited low levels of 16S rRNA gene sequence similarity (<92 %) with respect to all recognized species of the family Flavobacteriaceae, and formed a distinct phyletic line. In addition, several phenotypic properties readily served to differentiate the isolate from its phylogenetic neighbours (Table 1Go). On the basis of the data from this polyphasic study, strain JC2129T represents a novel genus and species of the family Flavobacteriaceae, for which the name Actibacter sediminis gen. nov., sp. nov. is proposed.

Description of Actibacter gen. nov.
Actibacter (Ac.ti.bac'ter. L. n. acta seaside; N.L. masc. n. bacter rod; N.L. masc. n. Actibacter rod from the seaside).

Cells are rod-shaped with rounded ends, non-flagellated and non-gliding. Gram-negative. Aerobic, chemoheterotrophic and mesophilic. Oxidase- and catalase-positive. Spores are not formed. Flexirubin-type pigments are absent. The major isoprenoid quinone is MK-6. The predominant cellular fatty acids are iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C13 : 0. As determined by 16S rRNA gene sequence analysis, the genus Actibacter is a member of the family Flavobacteriaceae. The type species is Actibacter sediminis.

Description of Actibacter sediminis sp. nov.
Actibacter sediminis (se.di.mi'nis. L. gen. n. sediminis of a sediment).

Cells are 0.7–0.9 µm wide and 2.4–3.2 µm long. Colonies on MA are circular, smooth, convex, entire and yellow-pigmented. Growth occurs at 5–45 °C (optimum, 37 °C), at pH 5–8 (optimum, pH 6) and in the presence of 1–10 % NaCl (optimum, 1–3 %) or 1–15 % sea salts (optimum, 1 %). Growth also occurs on R2A medium in the absence of NaCl and sea salt. Nitrate is not reduced to nitrite. Indole and H2S are not produced. Aesculin, gelatin, starch and Tween 80 are degraded, but DNA, agar, casein, crystalline cellulose (filter paper) and chitin are not. Alkaline phosphatase, esterase (C4), esterase lipase (C8), {alpha}-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and {alpha}-glucosidase activities are present, but lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, {alpha}-galactosidase, β-galactosidase, β-glucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, {alpha}-fucosidase and {alpha}-mannosidase activities are absent. Acid is not produced from glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate or phenylacetate. Growth occurs on peptone, tryptone and yeast extract. Acetate, citrate, pyruvate, sucrose and L-glutamate are utilized. Glycerol, L-leucine, L-proline, succinate, benzoate, L-proline and p-toluic acid are not utilized. The predominant cellular fatty acids are iso-C15 : 0 (19.8 %), iso-C15 : 1 G (14.0 %), iso-C17 : 0 3-OH (13.7 %) and iso-C13 : 0 (6.4 %). The DNA G+C content is 43–45 mol%.

The type strain, JC2129T (=KCTC 12704T =JCM 14002T), was isolated from sediment of getbol (a Korean word for tidal flat) on Ganghwa Island, South Korea.


   ACKNOWLEDGEMENTS
 
We thank Dr J. P. Euzéby for help with the nomenclature. This work was supported by a Korea Research Foundation grant (KRF-331-C00248) funded by the Korean Government (MOEHRD).


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