International Journal of Systematic and Evolutionary Microbiology (2015), 65, 485–490
DOI 10.1099/ijs.0.067132-0
Lutibacter oricola sp. nov., a marine bacterium isolated from seawater Hye-Ri Sung,1 Kee-Sun Shin1 and Sa-Youl Ghim2 Correspondence
1
Sa–Youl Ghim
2
[email protected]
Biological Resource Center, KRIBB, Daejeon 305-806, Republic of Korea School of Life Sciences, KNU Creative BioResearch Group (BK21 plus project), Institute for Microorganisms, Kyungpook National University, Daegu 702-701, Republic of Korea
A bacterial strain, UDC377T, was isolated from seawater samples collected at Seo-do on the coast of Dokdo island in the East Sea, and was subjected to taxonomic study using a polyphasic approach. Strain UDC377T was pale-yellow, Gram-staining-negative, non-motile, rod-shaped and aerobic. The strain grew optimally at 25–28 6C, in the presence of 2 % (w/v) NaCl and at pH 7.0–7.5. Strain UDC377T produced carotenoid pigments; however, it did not produce flexirubin-type pigments. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain UDC377T clustered with members of the genus Lutibacter and appeared most closely related to Lutibacter agarilyticus KYW566T (96.0 % 16S rRNA gene sequence similarity) followed by L. aestuarii MA-My1T (95.0 %), L. litoralis CL-TF09T (94.9 %), L. maritimus S7-2T (94.1 %) and L. flavus IMCC1507T (94.0 %). The DNA G+C content of strain UDC377T was 30.8 mol%. Strain UDC377T contained MK-6 as the predominant menaquinone, iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH as the major fatty acids, and phosphatidylethanolamine, two unknown aminolipids and six unknown lipids as the major polar lipids. Based on phenotypic properties and phylogenetic data presented, strain UDC377T is considered to represent a novel species of the genus Lutibacter, for which the name Lutibacter oricola sp. nov. is proposed. The type strain is UDC377T (5DSM 24956T5KCTC 23668T).
The genus Lutibacter in the family Flavobacteriaceae (Jooste, 1985) of the phylum Bacteroidetes (Garrity & Holt, 2001) was first proposed by Choi & Cho (2006) and, at the time of writing, comprises five species with validly published names. These five species, Lutibacter aestuarii (Lee et al., 2012), L. agarilyticus (Park et al., 2013) L. flavus (Choi et al., 2013), L. litoralis (Choi & Cho, 2006) and L. maritimus (Park et al., 2010), were all isolated from the western and southern coasts of the Korean peninsula. Species of the genus Lutibacter are Gram-staining-negative, aerobic, rod-shaped and yellowpigmented bacteria that contain menaquinone-6 (MK-6) as the major respiratory quinone and phosphatidylethanolamine (PE) and several unidentified lipids as the major polar lipids. During investigation of the biodiversity of marine bacteria from seawater, a novel pale-yellow bacterium was isolated. In the present study, we investigated the taxonomic position of this strain, designated UDC377T, using a polyphasic approach that included morphological, physiological, Abbreviations: FAME, fatty acid methyl ester; PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain UDC377T is HM031972. Two supplementary figures are available with the online Supplementary Material.
067132 G 2015 IUMS
Printed in Great Britain
biochemical and chemotaxonomic characterization, as well as phylogenetic analysis. In April 2008, strain UDC377T was isolated from a sample of seawater collected at Seo-do (37u 149 30.60 N 131u 59 54.60 E) on the coast of Dokdo island, in the East Sea. The strain was isolated on marine agar 2216 (MA; BD) (Yang et al., 2006), by means of the standard dilution plating technique and incubation at 25 uC for 7 days. The isolate was routinely incubated on MA, and preserved at 270 uC in marine broth (MB; BD) containing 15 % (v/v) glycerol. Cell morphology was investigated by light microscopy (Sw 804255; Samwon) and transmission electron microscopy (H-7100; Hitachi), using cells that had been grown on MA at 25 uC for 5 days. The presence of flagella was determined by using transmission electron microscopy on cells from an exponentially growing culture. Cell motility was investigated by observing cells grown in motility test agar (BBL; BD), and gliding motility was investigated using the hanging-drop method described by Bernardet et al. (2002). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to manufacturer’s instructions. Anaerobic growth was determined after incubation for 10 days at 25 uC in a GasPak anaerobic system (BBL) on MA and on MA supplemented with 0.1 % (w/v) potassium nitrate. The presence of flexirubin-type pigments was investigated by the 485
H.-R. Sung, K.-S. Shin and S.-Y. Ghim
bathochromatic shift test when a mass of bacteria collected on agar was flooded with 20 % KOH (Reichenbach, 1992). Carotenoid pigments were extracted from the biomass obtained from cultures grown for 2 days in MB at 25 uC with an acetone/methanol mixture (1 : 1, v/v), and then the absorption spectra were determined using a scanning UV/ visible spectrophotometer (UV-1650pc; Shimadzu). The temperature range for growth was tested on MA at 4, 10, 15, 20, 25, 28, 30, 32, 34, 37 and 40 uC. Growth at various concentration of NaCl (0, 0.5, 1, 1.5 and 2–8 %, in increments of 1.0 %) was investigated by supplementing the appropriate concentration of NaCl into MB prepared according to the formula of the BD medium except that NaCl was excluded. The pH range for growth was determined in MB adjusted to various pH values (pH 4.5–9.5, in increments of 0.5 pH units) by the addition of HCl or Na2CO3. Catalase activity was determined by bubble production in 3 % (w/v) H2O2 (Cowan & Steel, 1965) and oxidase activity was tested by using oxidase reagent (bioMe´rieux). Hydrolysis of casein, hypoxanthine, starch, tyrosine, xanthine and Tweens 20, 40, 60 and 80 was tested on MA, using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of CM-cellulose was investigated on MA containing 0.5 % (w/v) CM-cellulose (Sigma) and detected according to the method of Teather & Wood (1982). DNase activity was determined on DNase test agar (Difco) made with artificial seawater; the artificial seawater contained (l-1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates as sole carbon and energy sources was examined as described by Baumann & Baumann (1981) using medium supplemented with 2 % (v/v) Hutner’s mineral base and 1 % (v/v) vitamin solution (Staley, 1968). Acid production from carbohydrates was determined using the method of Leifson (1963). Susceptibility to antibiotics was determined by the disc diffusion method, using commercial antibiotic-impregnated discs (BBL; BD) containing the following (mg per disc unless otherwise stated): amikacin (30), ampicillin (10), carbenicillin (100), cephlothin (30), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), lincomycin (15), nalidixic acid (30), neomycin (30), novobiocin (30), penicillin G (10 IU), polymyxin B (300 IU), rifampicin (30), streptomycin (10), sulfamethoxazole (50), tetracycline (30), trimethoprim (5) and vancomycin (30). Enzyme activities and other biochemical tests were performed using API ZYM, API 20E and API 20NE systems (bioMe´rieux).
the amplified genes and subsequent phylogenetic analyses were performed as previously described (Yoon et al., 2003). DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Tamaoka & Komagata (1984), using a reverse-phase column. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and then analysed by HPLC (Shimadzu) and a reverse-phase column (YMC ODS-A, 15064.6 mm). For cellular fatty acid analysis, cell mass of strain UDC 377T, L. aestuarii KCTC 23499T, L. agarilyticus KCTC 23842T, L. flavus KACC 14312T, L. litoralis JCM 13034T, L. maritimus KCTC 22635T were harvested after cultivation for 3 days at 28 uC on MA. Fatty acid methyl esters (FAMEs) were saponified, methylated and extracted using the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). FAMEs were identified by using TSBA6 database of the MIDI (Sherlock Microbial Identification System, version 6.1). Polar lipids were extracted from freeze-dried cell materials according to Minnikin et al. (1984) and separated by 2D silica-gel thinlayer chromatography. Total lipids and specific functional groups were detected using molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol, as previously described (Minnikin et al., 1984; Komagata & Suzuki, 1987). The nearly complete 16S rRNA gene sequence of strain UDC377T (1459 nt) was determined. The EzTaxon-e server (http://www.ezbiocloud.net/eztaxon, Kim et al., 2012) was used to evaluate sequence similarity values between strain UDC377T and closely related taxa. Strain UDC377T showed the highest 16S rRNA gene sequence similarity to L. agarilyticus KYW566T (96.0 %) and other members of the genus Lutibacter (94.0–95.0 %). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain UDC377T fell within the clade of all species of the genus Lutibacter, which was supported by a bootstrap resampling value of 87 % (Fig. 1). The topology of maximum-likelihood and maximum-parsimony algorithms formed a robust clade, containing members of the genus Lutibacter. The detailed cultural, morphological, biochemical and physiological characteristics of strain UDC377T are given in the species description and Fig. S1 (available in the online Supplementary Material), and alongside comparative data on other species of the genus Lutibacter (Table 1).
For DNA extraction, isoprenoid quinone analysis and polar lipid analysis, cell biomass of strain UDC377T was obtained from cultures grown for 2 days in MB at 25 uC. Genomic DNA was extracted and purified as described by Yoon et al. (1996), except that RNase T1 was used in combination with RNase A to minimize RNA contamination. The 16S rRNA gene was amplified by PCR using two universal primers, as described by Yoon et al. (1998). Sequencing of
The predominant isoprenoid quinone detected in strain UDC377T was MK-6 in line with that observed in all species of the genus Lutibacter (Choi & Cho, 2006; Choi et al., 2013; Lee et al., 2012; Park et al., 2010, 2013) and all members of the family Flavobacteriaceae (Bernardet, 2011). The cellular fatty acid profiles of strain UDC377T and strains of all closely related species of the genus Lutibacter are shown in Table 2. The major fatty acids of the novel strain were iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH. The fatty acid profiles of strain UDC377T and type strains of five other species of the genus Lutibacter were mostly similar; however, there were some differences in the proportions of some fatty acids including C17 : 0 3-OH
486
International Journal of Systematic and Evolutionary Microbiology 65
Lutibacter oricola sp. nov.
Polaribacter butkevichii KMM 3938T (AY189722) 95/96/97
Polaribacter reichenbachii KMM 6386T (HQ853596)
90
Polaribacter irgensii 23-PT (M61002)
100/100/100
Polaribacter filamentus 215T (U73726)
100/100/100 76/83/81
Polaribacter franzmannii 301T (U14586)
Polaribacter dokdonensis DSW-5T (DQ004686)
84/71*
Polaribacter reichenbachii KMM 6386T (HQ891656) Polaribacter gangjinensis K17-16T (FJ425213)
93/87/79
Polaribacter porphyrae LNM-20T (AB695286) Tenacibaculum lutimaris TF-26T (AY661691) 99/97 Tenacibaculum aestuarii SMK-4T (DQ314760) /98 83
Tenacibaculum skagerrakense D30T (AF469612)
100/99/100
Tenacibaculum litoreum CL-TF13T (AY962294)
100/99/98
Tenacibaculum gallaicum A37.1T (AM746477)
Tenacibaculum maritimum NBRC 15946T (AB078057) Maritimimonas rapanae A31T (EU290161) Lutibacter aestuarii MA-My1T (HM234096) Lutibacter litoralis CL-TF09T (AY962293) Lutibacter flavus IMCC1507T (GU166749) 96/75/84
Lutibacter maritimus S7-2T (FJ598048)
Lutibacter agarilyticus KYW566T (JN864028)
87/79†
Lutibacter oricola UDC377T (HM031972) Namhaeicola litoreus DPG-25T (JN033800) 100
Actibacter sediminis JC2129T (EF67065) Aestuariicola saemankumensis SMK-142T (EU239499) 0.1
Flexibacter flexilis DSM 6793T (AB078050)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationship between strain UDC377T and members of the genus Lutibacter and other representatives of the family Flavobacteriaceae. Bootstrap values (.70 %) based on 1000 replications are shown at branch points for the neighbour-joining, the maximum-likelihood and maximum-parsimony methods, respectively. Filled circles indicate that the corresponding nodes was recovered by all treeing methods. Open circles indicate that the corresponding nodes were also recovered in the trees generated with neighbour-joining and maximumlikelihood methods. *Bootstrap values obtained from the neighbour-joining and maximum-likelihood algorithms, respectively; 3bootstrap values obtained from neighbour-joining and maximum-parsimony algorithms, respectively. Single bootstrap values obtained from neighbour-joining method. Flexibacter flexilis DSM 6793T (GenBank accession no. AB078050) was used as an outgroup. Bar, 0.1 substitutions per nucleotide position.
and iso-C14 : 0 3-OH. The DNA G+C content of strain UDC377T was 30.8 mol%, a value that lies within the range of DNA G+C contents reported for established species of http://ijs.sgmjournals.org
the genus Lutibacter (30.6–41.6 mol%). The polar lipids detected in strain UDC377T were PE, two unidentified aminolipids and six unidentified lipids (Fig. S2), which 487
H.-R. Sung, K.-S. Shin and S.-Y. Ghim
Table 1. Differential phenotypic characteristics of strain UDC377T and other members of the genus Lutibacter Strains: 1, UDC377T; 2, L. aestuarii KCTC 23499T; 3, L. agarilyticus KCTC 23842T; 4, L. flavus KACC 14312T; 5, L. litoralis JCM 13034T; 6, L. maritimus KCTC 22635T. Data are from this study unless indicated otherwise. All strains were positive for catalase activity; hydrolysis of aesculin and starch; enzyme activity of alkaline phosphatase, leucine arylamidase and b-galactosidase; resistance to amikacin, gentamicin, kanamycin, neomycin, streptomycin and tetracycline. All strains were negative for Gram-staining; activity of oxidase; production of flexirubin-type pigments, acetoin and H2S; citrate utilization; glucose fermentation; hydrolysis of CM-cellulose, hypoxanthine, urea and xanthine; enzyme activity of lipase, achymotrypsin, b-glucuronidase, a-glucosidase, a-mannosidase, a-fucosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase; resistance to carbenicillin, chloramphenicol, erythromycin, lincomycin and rifampicin. +, positive; 2, negative; W, weakly positive. Characteristic Optimal temperature for growth (uC) Growth at 37 uC Optimal pH for growth* Tolerance to 6 % NaCl* Anaerobic growth Hydrolysis of: Agarose DNA Gelatin Tyrosine Tween 20 Tween 40 Tween 60 Tween 80 Enzyme activity(API ZYM) Esterase Valine arylamidase Naphthol-AS-BI-phosphohydrolase b-Glucosidase N-Acetyl-b-glucosamidase API 20NE tests Reduction of nitrates to nitrites Indole production Resistant to: Ampicillin Cephlothin Nalidixic acid Novobiocin Penicillin G Polymyxin B Sulfamethoxazole Trimethoprim Vancomycin Carotenoid pigment (nm) DNA G+C contents (mol%)
1
2
3
4
5
6
25–28 2 7.0–7.5 2 2
30* +* 7.0–7.5* + +
25* 2* 7.0* 2 2
30* +* 8.0* 2 2
25–30* 2* 7.0–8.0* 2 2
25–30* +* 7.0–8.0* 2 +
+ + + + 2 2 2 +
+ + + + 2 2 2 2
2 2 2 + 2 2 + 2
2 + + 2 2 2 2 +
2 + 2 + 2 + 2 2
2 + + + + + + +
W
W
W
+
+
W
W
W
W
W
+
W
+
W
2 +
+
2 +
2
2 2
2 +
+ +
2 2
2
2
W
W
+ + + 2 2 + 2 2 2 450 30.8
+ 2 + 2 + + + 2 2 452a 30.6a
2 2 + + 2 2 2 + + 452, 470 41.6b
2 2 + 2 2 2 2 + 2 451, 478c 31.4c
2 2 + + 2 2 + 2 2 452, 478d 33.9d
+ + 2 + + + 2 2 2 450, 475e 34.6e
W W
+ 2 2
+ + 2
W
W
*Data from a, Lee et al. (2012), b, Park et al. (2013), c, Choi et al. (2013), d, Choi & Cho (2006), e, Park et al. (2010).
were similar to polar lipid profiles for members of the genus Lutibacter except for the presence of additional unidentified aminolipids and unidentified lipids (Choi et al., 2013, Lee et al., 2012). Based on phylogenetic and chemotaxonomic analyses, strain UDC377T was found to be a member of the genus Lutibacter. However, strain UDC377T was differentiated from other species of the
genus Lutibacter with regard to some phenotypic characteristics, including hydrolysis of macromolecules, enzyme activities and antibiotic resistance (Table 1). Based on phylogenetic, genomic, chemotaxonomic and phenotypic data, strain UDC377T represents a novel species of the genus Lutibacter, for which the name Lutibacter oricola sp. nov. is proposed.
488
International Journal of Systematic and Evolutionary Microbiology 65
Lutibacter oricola sp. nov.
Table 2. Cellular fatty acid composition (%) of strain UDC377T and type strains of species of the genus Lutibacter Strains: 1, UDC377T; 2, Lutibacter aestuarii KCTC 23499T; 3, L. agarilyticus KCTC 23842T; 4, L. flavus KACC 14312T; 5, L. litoralis JCM 13034T; 6, L. maritimus KCTC 22635T. All data were obtained in this study. Fatty acids that represented ,1 % of the total in all six strains are not shown. TR, Trace amount (,1 %); 2, not detected. Fatty acid
1
Straight-chain fatty acids C14 : 0 TR C16 : 0 1.2 Branched fatty acids TR iso-C13 : 0 iso-C14 : 0 5.3 14.3 iso-C15 : 0 iso-C16 : 0 2.5 4.6 iso-C15 : 1(G) 6.9 iso-C16 : 1(H) anteiso-C15 : 0 5.3 TR anteiso-C15 : 1 Unsaturated fatty acids 5.9 C15 : 1v6c C17 : 1v6c 1.7 Hydroxy fatty acids C15 : 0 2-OH 1.1 C17 : 0 2-OH TR C15 : 0 3-OH 4.9 2.9 C16 : 0 3-OH C17 : 0 3-OH 1.3 iso-C14 : 0 3-OH 2.1 iso-C15 : 0 3-OH 19.8 11.2 iso-C16 : 0 3-OH iso-C17 : 0 3-OH 2.9 Summed features* 1 2 3 1.9
2
3
4
5
6
TR
TR
TR
TR
1.1
1.3 1.6
TR
TR
1.9 10.3 28.3 1.7 6.0 3.0 2.2 2
TR
1.4 4.7 19.0
TR
TR
11.5 15.0 3.7 4.2 4.5 9.8
4.3 1.1
4.8 1.2
2 2 3.6
TR
TR
TR
2 1.0 13.8 11.4 4.2
2 1.4 6.5 18.6 4.4
1.5 1.4 1.0 1.8 2 2 24.9 5.1 2.6
1.7
1.2 4.9
2 1.2
TR
TR
1.7 TR
TR
11.1 2 16.6 1.8
TR
1.4 6.1 32.1 27.4 TR 2.5 6.7 4.9 TR 3.6 2.0 8.6 2 TR
1.7 2
5.4 3.8
TR
9.4
2
TR
TR
TR
2.5 2.6 4.2 TR 2 2 2 TR 16.8 10.5 4.7 10.1 7.4 4.2 TR
3.3
4.0
TR
*Summed features represent groups of two or more fatty acids that could not be separated using the MIDI system. Summed feature 1 contained C13 : 0 3-OH and/or iso-C15 : 1 I/H; summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
pH 7.0–7.5). Growth does not occur under anaerobic conditions on MA or on MA supplemented with 0.1 % (w/v) potassium nitrate. Cells are catalase-positive and oxidase-negative. Aesculin, DNA, gelatin, starch, Tween 80 and tyrosine are hydrolysed and agar is slightly hydrolysed, but casein, CM-cellulose, hypoxanthine, Tweens 20, 40 and 60, and xanthine are not. Acid production occurs in the presence of D-glucose, Dmannitol, D-mannose, maltose and sucrose. Weak acid production occurs with the addition of melibiose, but not with L-arabinose, cellobiose, D-fructose, D-galactose, myoinositol, lactose, melezitose, raffinose, L-rhamnose, Dribose, trehalose or D-xylose. Cellobiose, D-fructose, D-galactose, D-glucose, maltose, D-mannose, sucrose and D-xylose are utilized as sole carbon and energy sources. According to API 20NE tests, aesculin hydrolysis, gelatin liquefaction and PNPG (b-galactosidase) activities are positive, whereas nitrate reduction, glucose fermentation, arginine dihydrolase and urease activities are negative. In the API ZYM gallery, tests are positive for alkaline phosphatase, b-galactosidase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, weakly positive for esterase (C4), valine arylamidase and crystine arylamidase, and negative for esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosamidase, a-mannosidase and a-fucosidase activities. Susceptible to carbenicillin, chloramphenicol, erythromycin, lincomycin, novobiocin, penicillin G, rifampicin, sulfamethoxazole, trimethoprim and vancomycin, but resistant to amikacin, ampicillin, cephlothin, gentamicin, kanamycin, nalidixic acid, neomycin, polymyxin B, streptomycin and tetracycline. The predominant isoprenoid quinone is MK-6. The major polar lipids are PE, two unidentified aminolipids and six unidentified lipids. Major fatty acids (.10 % of total fatty acids) are iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH. The type strain UDC377T (5DSM 24956T5KCTC 23668T), was isolated from seawater collected at Seo-do, on Dokdo island, in the East Sea. The DNA G+C content of the type strain is 30.8 mol% (determined by HPLC).
Acknowledgements Description of Lutibacter oricola sp. nov. Lutibacter oricola (o.ri9co.la. L. fem. n. ora coast; L. suff. -cola (from L. n. incola), a dweller, inhabitant; N.L. masc. n. oricola an inhabitant of the coast). Cells are Gram-staining-negative, non-flagellated, nongliding and non-spore-forming rods (0.3–0.5 mm61.4– 2.3 mm). Cells grow well on MA but do not grow on TSA or R2A. Colonies on MA are circular, convex, smooth, slightly glistering pale-yellow and 2.5–3.0 mm in diameter after 5 days incubation at 25 uC, with ageing colonies turning slightly flat. Grows well on MA, with growth occurring at 8–32 uC (optimum 25–28 uC), with 0.5–5 % (w/v) NaCl (optimum 2 %) and at pH 5.5–8.5 (optimum, http://ijs.sgmjournals.org
This work was supported by a grant from NRF-2013M3A9A5076603 and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (2008-2004721).
References Baumann, P. & Baumann, L. (1981). The marine Gram staining negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer. Bernardet, J.-F. (2011). Family I. Flavobacteriaceae Reichenbach 1992.
In Bergey’s Manual of Systematic Bacteriology, vol. 4, pp. 106–111. Edited by N. R. Krieg, W. Ludwig, W. B. Whitman, B. P. Hedlund, 489
H.-R. Sung, K.-S. Shin and S.-Y. Ghim B. J. Paster, J. T. Staley, N. Ward, D. Brown & A. Parte. New York: Sprnger.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Bernardet, J.-F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002).
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Choi, D. H. & Cho, B. C. (2006). Lutibacter litoralis gen. nov., sp. nov.,
a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment. Int J Syst Evol Microbiol 56, 771–776. Choi, A., Yang, S.-J. & Cho, J.-C. (2013). Lutibacter flavus sp. nov., a
marine bacterium isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 946–951. Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of
Park, S., Kang, S.-J., Oh, T.-K. & Yoon, J.-H. (2010). Lutibacter
maritimus sp. nov., isolated from a tidal flat sediment. Int J Syst Evol Microbiol 60, 610–614. Park, S. C., Choe, H. N., Hwang, Y. M., Baik, K. S. & Seong, C. N. (2013). Lutibacter agarilyticus sp. nov., a marine bacterium isolated
from shallow coastal seawater. Int J Syst Evol Microbiol 63, 2678– 2683. Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
2nd edn, vol. 4, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new
prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942.
Medical Bacteria. London: Cambridge University Press.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Garrity, G. M. & Holt, J. G. (2001). The road map to the Manual. In
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 119– 166. Edited by D. R. Boone, R. W. Castenholtz & G. M. Garrity. New York: Springer.
Teather, R. M. & Wood, P. J. (1982). Use of Congo red-polysaccharide
interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43, 777– 780.
P. J. (1985). The taxonomy and significance of Flavobacterium–Cytophaga strains from dairy sources. PhD thesis, University of the Orange Free State.
Yang, S.-H., Kwon, K. K., Lee, H.-S. & Kim, S.-J. (2006). Shewanella
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
spongiae sp. nov., isolated from a marine sponge. Int J Syst Evol Microbiol 56, 2879–2882.
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccha-
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
romonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Lee, S.-Y., Lee, M.-H., Oh, T.-K. & Yoon, J.-H. (2012). Lutibacter
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
aestuarii sp. nov., isolated from a tidal flat sediment, and emended description of the genus Lutibacter Choi and Cho 2006. Int J Syst Evol Microbiol 62, 420–424.
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
Jooste,
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp.
marine bacteria. J Bacteriol 85, 1183–1184.
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
490
International Journal of Systematic and Evolutionary Microbiology 65
Leifson, E. (1963). Determination of carbohydrate metabolism of
DOI 10.1099/ijs.0.067132-0
Lutibacter oricola sp. nov., a marine bacterium isolated from seawater Hye-Ri Sung,1 Kee-Sun Shin1 and Sa-Youl Ghim2 Correspondence
1
Sa–Youl Ghim
2
[email protected]
Biological Resource Center, KRIBB, Daejeon 305-806, Republic of Korea School of Life Sciences, KNU Creative BioResearch Group (BK21 plus project), Institute for Microorganisms, Kyungpook National University, Daegu 702-701, Republic of Korea
A bacterial strain, UDC377T, was isolated from seawater samples collected at Seo-do on the coast of Dokdo island in the East Sea, and was subjected to taxonomic study using a polyphasic approach. Strain UDC377T was pale-yellow, Gram-staining-negative, non-motile, rod-shaped and aerobic. The strain grew optimally at 25–28 6C, in the presence of 2 % (w/v) NaCl and at pH 7.0–7.5. Strain UDC377T produced carotenoid pigments; however, it did not produce flexirubin-type pigments. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain UDC377T clustered with members of the genus Lutibacter and appeared most closely related to Lutibacter agarilyticus KYW566T (96.0 % 16S rRNA gene sequence similarity) followed by L. aestuarii MA-My1T (95.0 %), L. litoralis CL-TF09T (94.9 %), L. maritimus S7-2T (94.1 %) and L. flavus IMCC1507T (94.0 %). The DNA G+C content of strain UDC377T was 30.8 mol%. Strain UDC377T contained MK-6 as the predominant menaquinone, iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH as the major fatty acids, and phosphatidylethanolamine, two unknown aminolipids and six unknown lipids as the major polar lipids. Based on phenotypic properties and phylogenetic data presented, strain UDC377T is considered to represent a novel species of the genus Lutibacter, for which the name Lutibacter oricola sp. nov. is proposed. The type strain is UDC377T (5DSM 24956T5KCTC 23668T).
The genus Lutibacter in the family Flavobacteriaceae (Jooste, 1985) of the phylum Bacteroidetes (Garrity & Holt, 2001) was first proposed by Choi & Cho (2006) and, at the time of writing, comprises five species with validly published names. These five species, Lutibacter aestuarii (Lee et al., 2012), L. agarilyticus (Park et al., 2013) L. flavus (Choi et al., 2013), L. litoralis (Choi & Cho, 2006) and L. maritimus (Park et al., 2010), were all isolated from the western and southern coasts of the Korean peninsula. Species of the genus Lutibacter are Gram-staining-negative, aerobic, rod-shaped and yellowpigmented bacteria that contain menaquinone-6 (MK-6) as the major respiratory quinone and phosphatidylethanolamine (PE) and several unidentified lipids as the major polar lipids. During investigation of the biodiversity of marine bacteria from seawater, a novel pale-yellow bacterium was isolated. In the present study, we investigated the taxonomic position of this strain, designated UDC377T, using a polyphasic approach that included morphological, physiological, Abbreviations: FAME, fatty acid methyl ester; PE, phosphatidylethanolamine. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain UDC377T is HM031972. Two supplementary figures are available with the online Supplementary Material.
067132 G 2015 IUMS
Printed in Great Britain
biochemical and chemotaxonomic characterization, as well as phylogenetic analysis. In April 2008, strain UDC377T was isolated from a sample of seawater collected at Seo-do (37u 149 30.60 N 131u 59 54.60 E) on the coast of Dokdo island, in the East Sea. The strain was isolated on marine agar 2216 (MA; BD) (Yang et al., 2006), by means of the standard dilution plating technique and incubation at 25 uC for 7 days. The isolate was routinely incubated on MA, and preserved at 270 uC in marine broth (MB; BD) containing 15 % (v/v) glycerol. Cell morphology was investigated by light microscopy (Sw 804255; Samwon) and transmission electron microscopy (H-7100; Hitachi), using cells that had been grown on MA at 25 uC for 5 days. The presence of flagella was determined by using transmission electron microscopy on cells from an exponentially growing culture. Cell motility was investigated by observing cells grown in motility test agar (BBL; BD), and gliding motility was investigated using the hanging-drop method described by Bernardet et al. (2002). The Gram reaction was determined by using the bioMe´rieux Gram stain kit according to manufacturer’s instructions. Anaerobic growth was determined after incubation for 10 days at 25 uC in a GasPak anaerobic system (BBL) on MA and on MA supplemented with 0.1 % (w/v) potassium nitrate. The presence of flexirubin-type pigments was investigated by the 485
H.-R. Sung, K.-S. Shin and S.-Y. Ghim
bathochromatic shift test when a mass of bacteria collected on agar was flooded with 20 % KOH (Reichenbach, 1992). Carotenoid pigments were extracted from the biomass obtained from cultures grown for 2 days in MB at 25 uC with an acetone/methanol mixture (1 : 1, v/v), and then the absorption spectra were determined using a scanning UV/ visible spectrophotometer (UV-1650pc; Shimadzu). The temperature range for growth was tested on MA at 4, 10, 15, 20, 25, 28, 30, 32, 34, 37 and 40 uC. Growth at various concentration of NaCl (0, 0.5, 1, 1.5 and 2–8 %, in increments of 1.0 %) was investigated by supplementing the appropriate concentration of NaCl into MB prepared according to the formula of the BD medium except that NaCl was excluded. The pH range for growth was determined in MB adjusted to various pH values (pH 4.5–9.5, in increments of 0.5 pH units) by the addition of HCl or Na2CO3. Catalase activity was determined by bubble production in 3 % (w/v) H2O2 (Cowan & Steel, 1965) and oxidase activity was tested by using oxidase reagent (bioMe´rieux). Hydrolysis of casein, hypoxanthine, starch, tyrosine, xanthine and Tweens 20, 40, 60 and 80 was tested on MA, using the substrate concentrations described by Cowan & Steel (1965). Hydrolysis of CM-cellulose was investigated on MA containing 0.5 % (w/v) CM-cellulose (Sigma) and detected according to the method of Teather & Wood (1982). DNase activity was determined on DNase test agar (Difco) made with artificial seawater; the artificial seawater contained (l-1 distilled water): 23.6 g NaCl, 0.64 g KCl, 4.53 g MgCl2 . 6H2O, 5.94 g MgSO4 . 7H2O and 1.3 g CaCl2 . 2H2O (Bruns et al., 2001). Utilization of various substrates as sole carbon and energy sources was examined as described by Baumann & Baumann (1981) using medium supplemented with 2 % (v/v) Hutner’s mineral base and 1 % (v/v) vitamin solution (Staley, 1968). Acid production from carbohydrates was determined using the method of Leifson (1963). Susceptibility to antibiotics was determined by the disc diffusion method, using commercial antibiotic-impregnated discs (BBL; BD) containing the following (mg per disc unless otherwise stated): amikacin (30), ampicillin (10), carbenicillin (100), cephlothin (30), chloramphenicol (30), erythromycin (15), gentamicin (10), kanamycin (30), lincomycin (15), nalidixic acid (30), neomycin (30), novobiocin (30), penicillin G (10 IU), polymyxin B (300 IU), rifampicin (30), streptomycin (10), sulfamethoxazole (50), tetracycline (30), trimethoprim (5) and vancomycin (30). Enzyme activities and other biochemical tests were performed using API ZYM, API 20E and API 20NE systems (bioMe´rieux).
the amplified genes and subsequent phylogenetic analyses were performed as previously described (Yoon et al., 2003). DNA G+C content was determined by HPLC analysis of deoxyribonucleosides as described by Tamaoka & Komagata (1984), using a reverse-phase column. Isoprenoid quinones were extracted according to the method of Komagata & Suzuki (1987) and then analysed by HPLC (Shimadzu) and a reverse-phase column (YMC ODS-A, 15064.6 mm). For cellular fatty acid analysis, cell mass of strain UDC 377T, L. aestuarii KCTC 23499T, L. agarilyticus KCTC 23842T, L. flavus KACC 14312T, L. litoralis JCM 13034T, L. maritimus KCTC 22635T were harvested after cultivation for 3 days at 28 uC on MA. Fatty acid methyl esters (FAMEs) were saponified, methylated and extracted using the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). FAMEs were identified by using TSBA6 database of the MIDI (Sherlock Microbial Identification System, version 6.1). Polar lipids were extracted from freeze-dried cell materials according to Minnikin et al. (1984) and separated by 2D silica-gel thinlayer chromatography. Total lipids and specific functional groups were detected using molybdophosphoric acid, molybdenum blue, ninhydrin and a-naphthol, as previously described (Minnikin et al., 1984; Komagata & Suzuki, 1987). The nearly complete 16S rRNA gene sequence of strain UDC377T (1459 nt) was determined. The EzTaxon-e server (http://www.ezbiocloud.net/eztaxon, Kim et al., 2012) was used to evaluate sequence similarity values between strain UDC377T and closely related taxa. Strain UDC377T showed the highest 16S rRNA gene sequence similarity to L. agarilyticus KYW566T (96.0 %) and other members of the genus Lutibacter (94.0–95.0 %). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain UDC377T fell within the clade of all species of the genus Lutibacter, which was supported by a bootstrap resampling value of 87 % (Fig. 1). The topology of maximum-likelihood and maximum-parsimony algorithms formed a robust clade, containing members of the genus Lutibacter. The detailed cultural, morphological, biochemical and physiological characteristics of strain UDC377T are given in the species description and Fig. S1 (available in the online Supplementary Material), and alongside comparative data on other species of the genus Lutibacter (Table 1).
For DNA extraction, isoprenoid quinone analysis and polar lipid analysis, cell biomass of strain UDC377T was obtained from cultures grown for 2 days in MB at 25 uC. Genomic DNA was extracted and purified as described by Yoon et al. (1996), except that RNase T1 was used in combination with RNase A to minimize RNA contamination. The 16S rRNA gene was amplified by PCR using two universal primers, as described by Yoon et al. (1998). Sequencing of
The predominant isoprenoid quinone detected in strain UDC377T was MK-6 in line with that observed in all species of the genus Lutibacter (Choi & Cho, 2006; Choi et al., 2013; Lee et al., 2012; Park et al., 2010, 2013) and all members of the family Flavobacteriaceae (Bernardet, 2011). The cellular fatty acid profiles of strain UDC377T and strains of all closely related species of the genus Lutibacter are shown in Table 2. The major fatty acids of the novel strain were iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH. The fatty acid profiles of strain UDC377T and type strains of five other species of the genus Lutibacter were mostly similar; however, there were some differences in the proportions of some fatty acids including C17 : 0 3-OH
486
International Journal of Systematic and Evolutionary Microbiology 65
Lutibacter oricola sp. nov.
Polaribacter butkevichii KMM 3938T (AY189722) 95/96/97
Polaribacter reichenbachii KMM 6386T (HQ853596)
90
Polaribacter irgensii 23-PT (M61002)
100/100/100
Polaribacter filamentus 215T (U73726)
100/100/100 76/83/81
Polaribacter franzmannii 301T (U14586)
Polaribacter dokdonensis DSW-5T (DQ004686)
84/71*
Polaribacter reichenbachii KMM 6386T (HQ891656) Polaribacter gangjinensis K17-16T (FJ425213)
93/87/79
Polaribacter porphyrae LNM-20T (AB695286) Tenacibaculum lutimaris TF-26T (AY661691) 99/97 Tenacibaculum aestuarii SMK-4T (DQ314760) /98 83
Tenacibaculum skagerrakense D30T (AF469612)
100/99/100
Tenacibaculum litoreum CL-TF13T (AY962294)
100/99/98
Tenacibaculum gallaicum A37.1T (AM746477)
Tenacibaculum maritimum NBRC 15946T (AB078057) Maritimimonas rapanae A31T (EU290161) Lutibacter aestuarii MA-My1T (HM234096) Lutibacter litoralis CL-TF09T (AY962293) Lutibacter flavus IMCC1507T (GU166749) 96/75/84
Lutibacter maritimus S7-2T (FJ598048)
Lutibacter agarilyticus KYW566T (JN864028)
87/79†
Lutibacter oricola UDC377T (HM031972) Namhaeicola litoreus DPG-25T (JN033800) 100
Actibacter sediminis JC2129T (EF67065) Aestuariicola saemankumensis SMK-142T (EU239499) 0.1
Flexibacter flexilis DSM 6793T (AB078050)
Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences, showing the relationship between strain UDC377T and members of the genus Lutibacter and other representatives of the family Flavobacteriaceae. Bootstrap values (.70 %) based on 1000 replications are shown at branch points for the neighbour-joining, the maximum-likelihood and maximum-parsimony methods, respectively. Filled circles indicate that the corresponding nodes was recovered by all treeing methods. Open circles indicate that the corresponding nodes were also recovered in the trees generated with neighbour-joining and maximumlikelihood methods. *Bootstrap values obtained from the neighbour-joining and maximum-likelihood algorithms, respectively; 3bootstrap values obtained from neighbour-joining and maximum-parsimony algorithms, respectively. Single bootstrap values obtained from neighbour-joining method. Flexibacter flexilis DSM 6793T (GenBank accession no. AB078050) was used as an outgroup. Bar, 0.1 substitutions per nucleotide position.
and iso-C14 : 0 3-OH. The DNA G+C content of strain UDC377T was 30.8 mol%, a value that lies within the range of DNA G+C contents reported for established species of http://ijs.sgmjournals.org
the genus Lutibacter (30.6–41.6 mol%). The polar lipids detected in strain UDC377T were PE, two unidentified aminolipids and six unidentified lipids (Fig. S2), which 487
H.-R. Sung, K.-S. Shin and S.-Y. Ghim
Table 1. Differential phenotypic characteristics of strain UDC377T and other members of the genus Lutibacter Strains: 1, UDC377T; 2, L. aestuarii KCTC 23499T; 3, L. agarilyticus KCTC 23842T; 4, L. flavus KACC 14312T; 5, L. litoralis JCM 13034T; 6, L. maritimus KCTC 22635T. Data are from this study unless indicated otherwise. All strains were positive for catalase activity; hydrolysis of aesculin and starch; enzyme activity of alkaline phosphatase, leucine arylamidase and b-galactosidase; resistance to amikacin, gentamicin, kanamycin, neomycin, streptomycin and tetracycline. All strains were negative for Gram-staining; activity of oxidase; production of flexirubin-type pigments, acetoin and H2S; citrate utilization; glucose fermentation; hydrolysis of CM-cellulose, hypoxanthine, urea and xanthine; enzyme activity of lipase, achymotrypsin, b-glucuronidase, a-glucosidase, a-mannosidase, a-fucosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase; resistance to carbenicillin, chloramphenicol, erythromycin, lincomycin and rifampicin. +, positive; 2, negative; W, weakly positive. Characteristic Optimal temperature for growth (uC) Growth at 37 uC Optimal pH for growth* Tolerance to 6 % NaCl* Anaerobic growth Hydrolysis of: Agarose DNA Gelatin Tyrosine Tween 20 Tween 40 Tween 60 Tween 80 Enzyme activity(API ZYM) Esterase Valine arylamidase Naphthol-AS-BI-phosphohydrolase b-Glucosidase N-Acetyl-b-glucosamidase API 20NE tests Reduction of nitrates to nitrites Indole production Resistant to: Ampicillin Cephlothin Nalidixic acid Novobiocin Penicillin G Polymyxin B Sulfamethoxazole Trimethoprim Vancomycin Carotenoid pigment (nm) DNA G+C contents (mol%)
1
2
3
4
5
6
25–28 2 7.0–7.5 2 2
30* +* 7.0–7.5* + +
25* 2* 7.0* 2 2
30* +* 8.0* 2 2
25–30* 2* 7.0–8.0* 2 2
25–30* +* 7.0–8.0* 2 +
+ + + + 2 2 2 +
+ + + + 2 2 2 2
2 2 2 + 2 2 + 2
2 + + 2 2 2 2 +
2 + 2 + 2 + 2 2
2 + + + + + + +
W
W
W
+
+
W
W
W
W
W
+
W
+
W
2 +
+
2 +
2
2 2
2 +
+ +
2 2
2
2
W
W
+ + + 2 2 + 2 2 2 450 30.8
+ 2 + 2 + + + 2 2 452a 30.6a
2 2 + + 2 2 2 + + 452, 470 41.6b
2 2 + 2 2 2 2 + 2 451, 478c 31.4c
2 2 + + 2 2 + 2 2 452, 478d 33.9d
+ + 2 + + + 2 2 2 450, 475e 34.6e
W W
+ 2 2
+ + 2
W
W
*Data from a, Lee et al. (2012), b, Park et al. (2013), c, Choi et al. (2013), d, Choi & Cho (2006), e, Park et al. (2010).
were similar to polar lipid profiles for members of the genus Lutibacter except for the presence of additional unidentified aminolipids and unidentified lipids (Choi et al., 2013, Lee et al., 2012). Based on phylogenetic and chemotaxonomic analyses, strain UDC377T was found to be a member of the genus Lutibacter. However, strain UDC377T was differentiated from other species of the
genus Lutibacter with regard to some phenotypic characteristics, including hydrolysis of macromolecules, enzyme activities and antibiotic resistance (Table 1). Based on phylogenetic, genomic, chemotaxonomic and phenotypic data, strain UDC377T represents a novel species of the genus Lutibacter, for which the name Lutibacter oricola sp. nov. is proposed.
488
International Journal of Systematic and Evolutionary Microbiology 65
Lutibacter oricola sp. nov.
Table 2. Cellular fatty acid composition (%) of strain UDC377T and type strains of species of the genus Lutibacter Strains: 1, UDC377T; 2, Lutibacter aestuarii KCTC 23499T; 3, L. agarilyticus KCTC 23842T; 4, L. flavus KACC 14312T; 5, L. litoralis JCM 13034T; 6, L. maritimus KCTC 22635T. All data were obtained in this study. Fatty acids that represented ,1 % of the total in all six strains are not shown. TR, Trace amount (,1 %); 2, not detected. Fatty acid
1
Straight-chain fatty acids C14 : 0 TR C16 : 0 1.2 Branched fatty acids TR iso-C13 : 0 iso-C14 : 0 5.3 14.3 iso-C15 : 0 iso-C16 : 0 2.5 4.6 iso-C15 : 1(G) 6.9 iso-C16 : 1(H) anteiso-C15 : 0 5.3 TR anteiso-C15 : 1 Unsaturated fatty acids 5.9 C15 : 1v6c C17 : 1v6c 1.7 Hydroxy fatty acids C15 : 0 2-OH 1.1 C17 : 0 2-OH TR C15 : 0 3-OH 4.9 2.9 C16 : 0 3-OH C17 : 0 3-OH 1.3 iso-C14 : 0 3-OH 2.1 iso-C15 : 0 3-OH 19.8 11.2 iso-C16 : 0 3-OH iso-C17 : 0 3-OH 2.9 Summed features* 1 2 3 1.9
2
3
4
5
6
TR
TR
TR
TR
1.1
1.3 1.6
TR
TR
1.9 10.3 28.3 1.7 6.0 3.0 2.2 2
TR
1.4 4.7 19.0
TR
TR
11.5 15.0 3.7 4.2 4.5 9.8
4.3 1.1
4.8 1.2
2 2 3.6
TR
TR
TR
2 1.0 13.8 11.4 4.2
2 1.4 6.5 18.6 4.4
1.5 1.4 1.0 1.8 2 2 24.9 5.1 2.6
1.7
1.2 4.9
2 1.2
TR
TR
1.7 TR
TR
11.1 2 16.6 1.8
TR
1.4 6.1 32.1 27.4 TR 2.5 6.7 4.9 TR 3.6 2.0 8.6 2 TR
1.7 2
5.4 3.8
TR
9.4
2
TR
TR
TR
2.5 2.6 4.2 TR 2 2 2 TR 16.8 10.5 4.7 10.1 7.4 4.2 TR
3.3
4.0
TR
*Summed features represent groups of two or more fatty acids that could not be separated using the MIDI system. Summed feature 1 contained C13 : 0 3-OH and/or iso-C15 : 1 I/H; summed feature 3 contained C16 : 1v7c and/or C16 : 1v6c.
pH 7.0–7.5). Growth does not occur under anaerobic conditions on MA or on MA supplemented with 0.1 % (w/v) potassium nitrate. Cells are catalase-positive and oxidase-negative. Aesculin, DNA, gelatin, starch, Tween 80 and tyrosine are hydrolysed and agar is slightly hydrolysed, but casein, CM-cellulose, hypoxanthine, Tweens 20, 40 and 60, and xanthine are not. Acid production occurs in the presence of D-glucose, Dmannitol, D-mannose, maltose and sucrose. Weak acid production occurs with the addition of melibiose, but not with L-arabinose, cellobiose, D-fructose, D-galactose, myoinositol, lactose, melezitose, raffinose, L-rhamnose, Dribose, trehalose or D-xylose. Cellobiose, D-fructose, D-galactose, D-glucose, maltose, D-mannose, sucrose and D-xylose are utilized as sole carbon and energy sources. According to API 20NE tests, aesculin hydrolysis, gelatin liquefaction and PNPG (b-galactosidase) activities are positive, whereas nitrate reduction, glucose fermentation, arginine dihydrolase and urease activities are negative. In the API ZYM gallery, tests are positive for alkaline phosphatase, b-galactosidase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, weakly positive for esterase (C4), valine arylamidase and crystine arylamidase, and negative for esterase lipase (C8), lipase (C14), trypsin, a-chymotrypsin, a-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosamidase, a-mannosidase and a-fucosidase activities. Susceptible to carbenicillin, chloramphenicol, erythromycin, lincomycin, novobiocin, penicillin G, rifampicin, sulfamethoxazole, trimethoprim and vancomycin, but resistant to amikacin, ampicillin, cephlothin, gentamicin, kanamycin, nalidixic acid, neomycin, polymyxin B, streptomycin and tetracycline. The predominant isoprenoid quinone is MK-6. The major polar lipids are PE, two unidentified aminolipids and six unidentified lipids. Major fatty acids (.10 % of total fatty acids) are iso-C15 : 0 3-OH, iso-C15 : 0 and iso-C16 : 0 3-OH. The type strain UDC377T (5DSM 24956T5KCTC 23668T), was isolated from seawater collected at Seo-do, on Dokdo island, in the East Sea. The DNA G+C content of the type strain is 30.8 mol% (determined by HPLC).
Acknowledgements Description of Lutibacter oricola sp. nov. Lutibacter oricola (o.ri9co.la. L. fem. n. ora coast; L. suff. -cola (from L. n. incola), a dweller, inhabitant; N.L. masc. n. oricola an inhabitant of the coast). Cells are Gram-staining-negative, non-flagellated, nongliding and non-spore-forming rods (0.3–0.5 mm61.4– 2.3 mm). Cells grow well on MA but do not grow on TSA or R2A. Colonies on MA are circular, convex, smooth, slightly glistering pale-yellow and 2.5–3.0 mm in diameter after 5 days incubation at 25 uC, with ageing colonies turning slightly flat. Grows well on MA, with growth occurring at 8–32 uC (optimum 25–28 uC), with 0.5–5 % (w/v) NaCl (optimum 2 %) and at pH 5.5–8.5 (optimum, http://ijs.sgmjournals.org
This work was supported by a grant from NRF-2013M3A9A5076603 and a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (2008-2004721).
References Baumann, P. & Baumann, L. (1981). The marine Gram staining negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In The Prokaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Tru¨per, A. Balows & H. G. Schlegel. Berlin: Springer. Bernardet, J.-F. (2011). Family I. Flavobacteriaceae Reichenbach 1992.
In Bergey’s Manual of Systematic Bacteriology, vol. 4, pp. 106–111. Edited by N. R. Krieg, W. Ludwig, W. B. Whitman, B. P. Hedlund, 489
H.-R. Sung, K.-S. Shin and S.-Y. Ghim B. J. Paster, J. T. Staley, N. Ward, D. Brown & A. Parte. New York: Sprnger.
Minnikin, D. E., O’Donnell, A. G., Goodfellow, M., Alderson, G., Athalye, M., Schaal, A. & Parlett, J. H. (1984). An integrated
Bernardet, J.-F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002).
procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 2, 233–241.
Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, 1049–1070. Bruns, A., Rohde, M. & Berthe-Corti, L. (2001). Muricauda
ruestringensis gen. nov., sp. nov., a facultatively anaerobic, appendaged bacterium from German North Sea intertidal sediment. Int J Syst Evol Microbiol 51, 1997–2006. Choi, D. H. & Cho, B. C. (2006). Lutibacter litoralis gen. nov., sp. nov.,
a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment. Int J Syst Evol Microbiol 56, 771–776. Choi, A., Yang, S.-J. & Cho, J.-C. (2013). Lutibacter flavus sp. nov., a
marine bacterium isolated from a tidal flat sediment. Int J Syst Evol Microbiol 63, 946–951. Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of
Park, S., Kang, S.-J., Oh, T.-K. & Yoon, J.-H. (2010). Lutibacter
maritimus sp. nov., isolated from a tidal flat sediment. Int J Syst Evol Microbiol 60, 610–614. Park, S. C., Choe, H. N., Hwang, Y. M., Baik, K. S. & Seong, C. N. (2013). Lutibacter agarilyticus sp. nov., a marine bacterium isolated
from shallow coastal seawater. Int J Syst Evol Microbiol 63, 2678– 2683. Reichenbach, H. (1992). The order Cytophagales. In The Prokaryotes,
2nd edn, vol. 4, pp. 3631–3675. Edited by A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer. Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI Inc. Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new
prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942.
Medical Bacteria. London: Cambridge University Press.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Garrity, G. M. & Holt, J. G. (2001). The road map to the Manual. In
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 1, pp. 119– 166. Edited by D. R. Boone, R. W. Castenholtz & G. M. Garrity. New York: Springer.
Teather, R. M. & Wood, P. J. (1982). Use of Congo red-polysaccharide
interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43, 777– 780.
P. J. (1985). The taxonomy and significance of Flavobacterium–Cytophaga strains from dairy sources. PhD thesis, University of the Orange Free State.
Yang, S.-H., Kwon, K. K., Lee, H.-S. & Kim, S.-J. (2006). Shewanella
Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e:
spongiae sp. nov., isolated from a marine sponge. Int J Syst Evol Microbiol 56, 2879–2882.
a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, 716–721.
Yoon, J.-H., Kim, H., Kim, S.-B., Kim, H.-J., Kim, W. Y., Lee, S. T., Goodfellow, M. & Park, Y.-H. (1996). Identification of Saccha-
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
romonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Bacteriol 46, 502–505.
Lee, S.-Y., Lee, M.-H., Oh, T.-K. & Yoon, J.-H. (2012). Lutibacter
Yoon, J.-H., Lee, S. T. & Park, Y.-H. (1998). Inter- and intraspecific
aestuarii sp. nov., isolated from a tidal flat sediment, and emended description of the genus Lutibacter Choi and Cho 2006. Int J Syst Evol Microbiol 62, 420–424.
phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences. Int J Syst Bacteriol 48, 187–194.
Jooste,
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp.
marine bacteria. J Bacteriol 85, 1183–1184.
nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449–454.
490
International Journal of Systematic and Evolutionary Microbiology 65
Leifson, E. (1963). Determination of carbohydrate metabolism of
