Euzebyella algicola sp . nov . , a marine bacterium of the family Flavobacteriaceae , isolated from green algae

A Gram-negative, yellow-pigmented, non-flagellated, gliding, rod-shaped and aerobic bacterium, designated MEBiC 12267, was isolated from green algae of Jeju Island. 16S rRNA gene sequence analysis revealed that the strain MEBiC 12267 was affiliated to the genus Euzebyella of the family Flavobacteriaceae and showed the highest similarity to Euzebyella marina KCTC 42440 (98.5%). The DNA–DNA relatedness value of strain MEBiC 12267 with E. marina KCTC 42440 was 25%. Growth was observed at 10–37 C (optimum, 30–33 C), at pH 6.0–9.5 (optimum, 8.0–8.5) and with 0.5–9.0% (w/v) NaCl (optimum, 2.5– 3.5%). The predominant cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The major respiratory quinone was MK-6. Polar lipids included phosphatidylethanolamine, seven unidentified lipids and two unidentified aminolipids. The DNA G+C content was 40.7mol%. On the basis of the data from the polyphasic taxonomic study, it was concluded that the strain MEBiC 12267 represents a novel species within the genus Euzebyella, for which the name Euzebyella algicola sp. nov. is proposed. The type strain of E. algicola is MEBiC 12267 (=KCCM 43264=JCM 32170). The family Flavobacteriaceae is one of the major branches of the phylum Bacteroidetes [1] and was first proposed by Jooste [2]. Subsequently, the name of the family was validly published by Reichenbach [3] and emended by Bernardet et al. [4, 5]. The genus Euzebyella, a member of the family Flavobacteriaceae, was first proposed by Lucena et al. [6]. The genus Euzebyella comprises two species with validly published names: Euzebyella saccharophila KCTC 22655, isolated from the western Mediterranean Sea [6], and Euzebyella marina KCTC 42440, isolated from seawater of the Yellow Sea [7]. In the present study, a novel bacterial strain, MEBiC 12267, is characterized using a polyphasic approach to determine its taxonomic position. Based on the collective findings, we propose that this strain is a new species of the genus Euzebyella. Strain MEBiC 12267 was isolated from a sample of the green algae Ulva species collected from a coastal region of Jeju Island, Republic of Korea (33 30¢ 27† N, 126 53¢ 46† E) on 6 October 2015. For strain isolation, a small piece of algal frond was homogenized, diluted with sterile seawater and spread onto marine agar 2216 (MA; Difco). Inoculated plates were incubated at 25 C for 5 days and then individual colonies were isolated from MA on the basis of morphological differences. After primary isolation and purification, the strain was routinely cultured on marine broth 2216 (MB; Difco) at 30 C and preserved with 20% (v/v) glycerol at 80 C. The type strains E. marina KCTC 42440 and E. saccharophila KCTC 22655 were used as reference strains for phenotypic tests and grown on MA at 30 C. Cell morphology and the presence of flagella were examined using a transmission electron microscope (Tecnai G Spirit Twin, FEI) after negative staining with cells grown on MA for 2 days. Gram staining was determined using the Gram stain kits (BD) according to the manufacturer’s instructions. Gliding motility was determined using the hanging drop technique as described by Bernardet et al. [5]. The growth temperature was tested on MA incubated at 4, 10, 15 C (5 days) and 20, 28, 30, 33, 37, 40 C (48 h). Growth with 0– 5%NaCl (at intervals of 0.5%, w/v) and 6–16% (at intervals of 1%, w/v) was performed in NP broth (containing 0.5% Author affiliations: Department of Applied Research, National Marine Biodiversity Institute of Korea, 75, Jangsan-ro 101beon-gil, Seocheon-gun, Chungcheongnam-do, 33662, Republic of Korea; Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea, 75, Jangsan-ro 101beon-gil, Seocheon-gun, Chungcheongnam-do, 33662, Republic of Korea; Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, 787 Haeanro, Ansan 15627, Republic of Korea; Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea; National Marine Biodiversity Institute of Korea, Seocheon 33662, Republic of Korea. *Correspondence: Sang-Jin Kim, s-jkim@kiost.ac.kr or s-jkim@mabik.re.kr

The family Flavobacteriaceae is one of the major branches of the phylum Bacteroidetes [1] and was first proposed by Jooste [2].Subsequently, the name of the family was validly published by Reichenbach [3] and emended by Bernardet et al. [4,5].The genus Euzebyella, a member of the family Flavobacteriaceae, was first proposed by Lucena et al. [6].The genus Euzebyella comprises two species with validly published names: Euzebyella saccharophila KCTC 22655 T , isolated from the western Mediterranean Sea [6], and Euzebyella marina KCTC 42440 T , isolated from seawater of the Yellow Sea [7].In the present study, a novel bacterial strain, MEBiC 12267 T , is characterized using a polyphasic approach to determine its taxonomic position.Based on the collective findings, we propose that this strain is a new species of the genus Euzebyella.
Strain MEBiC 12267 T was isolated from a sample of the green algae Ulva species collected from a coastal region of Jeju Island, Republic of Korea (33 30¢ 27 † N, 126 53¢ 46 † E) on 6 October 2015.For strain isolation, a small piece of algal frond was homogenized, diluted with sterile seawater and spread onto marine agar 2216 (MA; Difco).Inoculated plates were incubated at 25 C for 5 days and then individual colonies were isolated from MA on the basis of morphological differences.After primary isolation and purification, the strain was routinely cultured on marine broth 2216 (MB; Difco) at 30 C and preserved with 20 % (v/v) glycerol at À80 C. The type strains E. marina KCTC 42440 T and E. saccharophila KCTC 22655 T were used as reference strains for phenotypic tests and grown on MA at 30 C.
Cell morphology and the presence of flagella were examined using a transmission electron microscope (Tecnai G 2 Spirit Twin, FEI) after negative staining with cells grown on MA for 2 days.Gram staining was determined using the Gram stain kits (BD) according to the manufacturer's instructions.Gliding motility was determined using the hanging drop technique as described by Bernardet et al. [5].The growth temperature was tested on MA incubated at 4, 10, 15 C (5 days) and 20, 28, 30, 33, 37, 40 C (48 h).Growth with 0-5 % NaCl (at intervals of 0.5 %, w/v) and 6-16 % (at intervals of 1 %, w/v) was performed in NP broth (containing 0. Oxidase and catalase activities were determined by using the oxidase reagent (bioM erieux) and by the production of bubbles after the addition of a drop of 3 % H 2 O 2 solution, respectively.Hydrolysis of starch, DNA, casein, and Tweens 40, 60 and 80 was tested after 5 days incubation on MAbased media.Carbon utilization was determined using Biolog GN2 plate according to the manufacturer's instructions with artificial sea water (ASW, containing 2.8 % NaCl, 0.5 % MgCl 2 , 0.2 % MgSO 4 , 0.1 % CaCl 2 , 0.1 % KCl, 0.0001 % FeSO 4 and distilled water; [8]) as the cell suspension solution at 30 C for 2 days.Acid production from carbohydrates were determined using API 50 CH strips (bioM erieux) according to the manufacturer's instructions except that the API 50 CHB/E medium for the cell suspension was supplemented with 3 % NaCl, 1 % MgCl 2 Á6H 2 O and 0.1 % CaCl 2 .The enzymatic activities and biochemical properties were investigated by using API 20NE and API ZYM strips (bioM erieux) according to the manufacturer's instructions.Inoculum was the cell suspension in ASW solution and it was incubated at 30 C for 2 days.Antibiotic susceptibility was performed using the disc-diffusion method (Neosensitabs; Rosco) on MA and the growth inhibition zones were observed after 3 days incubation at 30 C.
The fatty acid methyl esters in whole cells of strains MEBiC 12267 T , and two reference strains, E. marina KCTC 42440 T and E. saccharophila KCTC 22655 T , grown on MA at 30 C for 2 days were analysed by gas chromatography (Agilent technologies 7890B) according to the instructions of the Microbial Identification System (MIDI; version 6.3) with the RTSBA6 database.The DNA G+C content and isoprenoid quinone composition were analysed by using reversedphase high-performance liquid chromatography [9] after incubation on MA at 30 C for 2 days.Polar lipids were examined by two-dimensional thin-layer chromatography after incubation on MA at 30 C for 2 days and identified using the procedures described by Minnikin et al. [10].
Extraction of genomic DNA was performed by using Exgene DNA extraction kit (gene All), and the amplified 16S rRNA gene sequencing was performed with an Applied Biosystems automated sequencer (ABI 3730XL) at Macrogen (Seoul, Republic of Korea).To ascertain the phylogenetic position of strain MEBiC 12267 T , the 16S rRNA gene sequence was compared with validly published species from the EzTaxon server (old.ezbiocloud.net/;[11]).A total of 1352 unambiguously aligned sequences were compared and phylogenetic trees were reconstructed using the neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms in the MEGA 5 [12].The resulting tree topologies were evaluated using bootstrap analyses based on 1000 resamplings.Analysis of DNA-DNA relatedness was also carried out using dot-blot hybridization [13].
Comparative analysis of the 16S rRNA gene sequences showed that strain MEBiC 12267 T was closely related to E. marina KCTC 42440 T with a similarity of 98.5 %, followed by E. saccharophila KCTC 22655 T (97.7 %).Phylogenetic analysis based on 16S rRNA gene sequences using the NJ, ML and MP algorithms showed that strain MEBiC 12267 T formed a monophyletic clade with other members of the genus Euzebyella (Fig. 1).Consequently, sequence comparisons based on almost-complete 16S rRNA gene sequences showed a clear affiliation of the isolate to the genus Euzebyella.The DNA-DNA relatedness value between the isolate and E. marina KCTC 42440 T , which showed the highest similarity (98.5 %), was 25 %, which was clearly below the 70 % threshold generally accepted for species delineation [14].
Cells of strain MEBiC 12267 T were Gram-negative, aerobic and non-flagellated (Fig. S1, available in the online version of this article) but showed gliding motility.The novel isolate was resistant to gentamicin (10 µg), kanamycin (30 µg), penicillin (10 U), streptomycin (10 µg) and tetracyclines (30 µg) and susceptible to ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), nalidixan (30 µg), rifampicin (5 µg) and vancomycin (30 µg).Strain MEBiC 12267 T did not grow without NaCl or in TY broth supplemented with only NaCl or MgCl 2 or CaCl 2 , but showed slight growth when both NaCl and MgCl 2 were added to the medium and showed good growth when NaCl, MgCl 2 and CaCl 2 were added to TY broth.Further morphological, physiological and biochemical characteristics of the isolate are given in the species description and Table 1 along with those of two reference strains, E. marina KCTC 42440 T and E. saccharophila KCTC 22655 T .
16S rRNA gene sequence analysis suggested that strain MEBiC 12267 T belonged to the genus Euzebyella, and its main chemotaxonomic features also corresponded to those of the genus.DNA-DNA relatedness among strain MEBiC 12267 T and E. marina KCTC 42440 T was 25 %.Physiological and biochemical tests distinguished strain MEBiC 12267 T from related species of the genus (Table 1).Therefore, strain MEBiC 12267 T should be classified as representative of a novel species of the genus Euzebyella, for which the name Euzebyella algicola sp.nov. is proposed.
The type strain is MEBiC 12267 T (=KCCM 43264 T =JCM 32170 T ), isolated from a sample of the green algae Ulva species collected from a coastal region of Jeju Island, Republic of Korea.

Fig. 1 .
Fig. 1.Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain MEBiC 12267 T (in bold type) and related taxa.GenBank accession numbers are given in parentheses.Bootstrap values (>50 %) based on 1000 replicates are indicated at nodes.Closed and open circles indicate nodes recovered from the three treeing methods (neighbour-joining, maximum-likelihood and maximum-parsimony) or two treeing methods, respectively.Nonlabens antarcticus AKS622 T was used as an outgroup.Bar, 0.01 changes per nucleotide position.