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J. Microbiol. Biotechnol. (2013), 23(11), 1509–1518 http://dx.doi.org/10.4014/jmb.1308.08007 Research Article jmb

Isolation and Characterization of a Novel Agar-Degrading Marine Bacterium, Gayadomonas joobiniege gen, nov, sp. nov., from the Southern Sea, Korea Won-Jae Chi1, Jae-Seon Park1, Min-Jung Kwak2, Jihyun F. Kim3, Yong-Keun Chang4, and Soon-Kwang Hong1*

1Division of Biological Science and Bioinformatics, Myongji University, Yongin 449-728, Republic of Korea 2Biosystems and Bioengineering Program, University of Science and Technology, Daejeon 305-350, Republic of Korea 3Department of Systems Biology, Yonsei University, Seoul 120-749, Republic of Korea 4Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea

Received: August 2, 2013 Revised: August 14, 2013 An agar-degrading bacterium, designated as strain G7T, was isolated from a coastal seawater Accepted: August 20, 2013 sample from Gaya Island (Gayado in Korean), Republic of Korea. The isolated strain G7T is gram-negative, rod shaped, aerobic, non-motile, and non-pigmented. A similarity search based on its 16S rRNA gene sequence revealed that it shares 95.5%, 90.6%, and 90.0% T First published online similarity with the 16S rRNA gene sequences of YM01, August 22, 2013 sagamiensis, and pacifica W3-3AT, respectively. Phylogenetic analyses demonstrated T *Corresponding author that strain G7 formed a distinct monophyletic clade closely related to of the family Phone: +82-31-330-6198; in the -like . The G+C content of strain Fax: +82-31-335-8249; G7T was 41.12 mol%. The DNA-DNA hybridization value between strain G7T and the E-mail: [email protected] phylogenetically closest strain YM01T was 19.63%. The genomes of G7T and YM01T had an average ANIb value of 70.00%. The predominant isoprenoid quinone of this particular strain was ubiquinone-8, whereas that of C. agarivorans YM01T was menaquinone-7. The major fatty T acids of strain G7 were Iso-C15:0 (41.47%), Anteiso-C15:0 (22.99%), and C16:1ω7c/iso-C15:0 2-OH (8.85%), which were quite different from those of YM01T. Comparison of the phenotypic characteristics related to carbon utilization, enzyme production, and susceptibility to antibiotics also demonstrated that strain G7T is distinct from C. agarivorans YM01T. Based on its phenotypic, chemotaxonomic, and phylogenetic distinctiveness, strain G7T was considered a novel genus and species in the Gammaproteobacteria, for which the name Gayadomonas joobiniege gen. nov. sp. nov. (ATCC BAA-2321 = DSM25250T = KCTC23721T) is proposed. pISSN 1017-7825, eISSN 1738-8872

Copyright© 2013 by Keywords: Agarase, Alteromonadaceae, Alteromonas-like Gammaproteobacteria, agar degradation, The Korean Society for Microbiology Gayadomonas joobiniege and Biotechnology

Introduction antioxidant activity, therapeutic activity in inflammatory disease, and antitumor activity, etc. [3, 11, 19], which may Marine microorganisms have received a great deal of broaden its application in the food, cosmetic, and attention in various efforts to utilize the abundant biological pharmaceutical industries as well as biorefinement or resources in the sea. One of these resources is agar, which biofuel industries. is a major component of red algae [6]. Agar is widely used For an efficient hydrolysis of agar, research seeking a as a dietary ingredient in food and as a gelling agent in good agarase has been under intense spotlight. A number solid culture media for microbial growth. Recently, agar of microorganisms with agar-hydrolyzing activity, such as oligosaccharides have also been reported to have marinoglutinosa [30], Alteromonas atlantica

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[1], mesophila [31], Catenovulum agarivorans [37], and Thalassomonas agarivorans [18], which are classified as Alteromonas-like Gammaproteobacteria [14, 16], have been isolated from marine environments. We recently isolated another Alteromonas-like marine bacterium (designated as strain G7T), which can grow on minimal medium supplemented with agar as the sole carbon source at higher temperature (above 40oC). Genomic sequencing of G7T revealed that it had many genes encoding hydrolytic enzymes: 50 sulfatases, 17 glycoside hydrolases, 13 agarases, 8 β-galactosidases, 3 altronate hydrolases, and 1 cellulase, which may act in the complete hydrolysis of sulfated algal polysaccharides [24]. Because of the great potential for the bacterium to be used as a bioresource for bioconversion of algal polysaccharides, we used a polyphasic taxonomic approach for the classification of strain G7T. This report describes the characteristics of the G7T strain as a novel genus of the family Alteromonadaceae.

Materials and Methods

Chemicals Agar and agarose were purchased from Amresco Inc. (USA) and Takara Shuzo Inc. (Japan), respectively. All other chemicals were purchased from Sigma Chemical Co. (USA). Fig. 1. Agarase production by strain G7T. (A) Detection of agarolytic activity of strain G7T on agar plate. The Isolation of Agarase-Producing Microorganisms strain was cultured on an ASW-YP agar plate at 40oC for 2 days (left), Coastal seawater of Gaya Island, Republic of Korea, was and Lugol’s iodine solution was overlaid to detect reducing sugars collected to isolate agar-degrading . The collected sample and degraded product from agar by agarase (right). (B) Cell growth was serially diluted from 10-1 to 10-5, and 200 µl of each dilution and agarase production of strain G7T in ASW-YP broth depending on was smeared on an artificial seawater (ASW) agar plate [20] cultivation time. The agarolytic activity was estimated by the colorimetric containing 1.0% yeast extract (w/v) and 0.3% bacto peptone (w/v) DNS method to measure the reducing sugar that resulted from (ASW-YP), and was incubated aerobically at 40oC for 24 h. The hydrolysis of agar as the substrate. Each value is an average of three

● ● ■ ■ ASW contained 6.1 g Tris base, 12.3 g MgSO4, 0.74 g KCl, 0.13 g parallel replicates. - , Cell growth; - , agarase activity.

(NH4)2HPO4, 17.5 g NaCl, and 0.14 g CaCl2 dissolved in 1 L of distilled water. A total of 1,136 colonies were collected and o transferred to fresh ASW-YP plates and incubated at 40 C for 24 h. at regular time intervals, and the optical density (OD) was The plate was stained with Lugol’s iodine solution (0.05 M iodine measured at 600 nm (OD600) to plot the growth curve. The sample in 0.12 M KI) to detect agarase activity (Fig. 1A). Colonies with was centrifuged at 14,000 rpm for 10 min and its supernatant was high agarase activity were isolated from the replica plate and collected to measure agarase activity. A substrate solution containing transferred to a fresh ASW-YP plate. The bacterial colonies were 0.2% agarose in 10 mM Tris-HCl (pH 8.0) was used for the agarase streaked five times to obtain a single colony for pure culture. One reaction. Agarase activity was measured by the previously marine bacterium with agar-hydrolyzing activity was selected described method [27] using 3,5-dinitrosalicylic acid (DNS). One T and designated as strain G7 in this study. After incubation in unit (U) of agarase was defined as the amount of enzyme that o ASW-YP broth at 40 C for 1 day, the culture was supplemented produced 1 µmol galactose per minute at 40oC. Galactose was used o with 10% glycerol (w/v) and stored at -80 C as the stock culture. as a reference reducing sugar for preparing the standard curve.

T Production of Agarase by Strain G7 16S rRNA Sequencing and Construction of Phylogenetic Tree T Cell growth and agarase activity of strain G7 were observed by The bacterial strain was cultured in ASW-YP liquid medium for incubating it in ASW-YP liquid medium containing 0.1% agar (w/v) 3 days, and genomic DNA was extracted with a genomic DNA o at 40 C for 72 h (Fig. 1B). Then, 1 ml of culture broth was sampled extraction kit (Promega Co., USA). The 16S rRNA gene was

J. Microbiol. Biotechnol. Gayadomonas joobiniege gen. nov. sp. nov. 1511

amplified by PCR using universal bacterial primers (27F, 5’- To determine antibiotic susceptibility, strain G7T and C. agarivorans AGAGTTTGATCCTGGCTCAG-3’; and 1492R, 5’-TACCTTGTT YM01 were smeared on ASW-YP agar plates and incubated at ACGACTT-3’), and nucleotide sequencing was performed using 40oC for 1 h. Paper discs containing 30 µl of thiostrepton (100 µg/ml), an Applied Biosystems 3730xl DNA Analyzer. Analysis of the 16S kanamycin (100 µg/ml), neomycin (100 µg/ml), ampicillin (100 µg/ml), rRNA gene sequence revealed that strain G7T belongs to the class apramycin (100 µg/ml), and chloramphenicol (100 µg/ml) were Gammaproteobacteria. The 16S rRNA gene sequences of type strains placed on the smear plate. The plates were incubated at 40oC for related to strain G7T were collected from the EzTaxon server 24 h and the zone of inhibition surrounding each antibiotic disc (http://www.eztaxon.org; [9]). The 16S rRNA sequences were was measured. aligned using ClustalW software [35], and the 5’ and 3’ gaps were edited using the BioEdit program [13]. Neighbor-joining (NJ), Chemotaxonomic Characteristics maximum likelihood (ML), and maximum parsimony (MP) methods The major respiratory quinones of strain G7T and C. agarivorans from the PHYLIP suite program [12] were used to construct the YM01 were analyzed by reverse-phase high-performance liquid phylogenetic tree. The bootstrap value was calculated using data chromatography (HPLC) after growth on Marine Broth (MB; Difco) restructured nearly 1,000 times and marked into the branching plates [23]. Cellular fatty acid methyl ester (FAME) mixtures were point. The evolutionary distance matrix was estimated according prepared from G7T and YM01 cells grown on MB (Difco) plates for to Kimura’s 2-parameter model [21]. 4 days by methyl esterification [28] and analyzed by gas chromatography using the Microbial Identification software package [32]. DNA-DNA Hybridization Genomic DNAs from strain G7T and the phylogenetically Results and Discussion closest type strain C. agarivorans YM01 were used for DNA-DNA T hybridization. Escherichia coli KCCM12119 was used as a negative Phenotypic Characteristics of the Agar-Hydrolyzing Isolate, control. DNA probe preparation and the hybridization reaction G7T were performed with the DIG High Prime DNA Labeling and Strain G7T is a gram-negative, rod-shaped bacterium. It Detection Starter Kit II (Roche Applied Science, Germany) according forms non-pigmented colonies that are circular and smooth to the manufacturer’s instruction. The resulting hybridization and produces agarases (Fig. 1A). Strain G7T produces signals were measured using the Quantity One Program (Bio-Rad, USA). The signal from strain G7T was set at 100%. colonies that are approximately 1.5 mm in diameter after incubation on an ASW-YP agar plate at 40oC for 48 h. The Phenotypic and Biochemical Characteristics growth of strain G7T in ASW-YP broth containing 0.1% Bacterial cells were grown on ASW-YP agar plates for 2 days and agar as the sole carbon source started to increase from 3 h, their morphology, size, and motility were examined by phase- reached its maximum level (OD600 = 0.85) at 36 h, and was contrast microscopy with a BX51 microscope (Olympus, USA). Cells were gram stained using a gram stain kit (BD, USA) according to the manufacturer’s instructions. Flagella were observed by transmission electron microscopy (TEM) after negative staining with 1% (w/v) phosphotungstic acid. Growth under anaerobic conditions was examined in an anaerobic jar system (GasPak system; BBL, USA). Biochemical tests of strain G7T and C. agarivorans YM01 were performed using the API ZYM strips (bioMérieux, France) according to the manufacturer’s instructions, with the exception that the bacterial suspension was prepared in ASW broth. Utilization of carbon sources were tested in 25 ml of ASW broth containing various carbon sources (0.2%) in 100 ml baffled flask by incubating the inoculated flasks at 28oC for 5 days with vigorous shaking (170 rpm). The NaCl requirement was determined by growth in ASW-YP medium containing 0%, 0.5%, 1%, 2%, 3%, 5%, 6%, 7%, 8%, 10%, 15%, and 20% NaCl (w/v). Strain G7T was inoculated in ASW-YP medium at pH 4, 5, 6, 7, 8, 9, and 10, and o incubated at 40 C for 3 days to investigate the effect of initial pH Fig. 2. Transmission electron microscopy (TEM) of strain G7T. on growth. The isolate was inoculated on ASW-YP agar plates and The strain was grown on an ASW-YP plate at 40oC for 2 days and was o o o o o o incubated at 4 C, 15 C, 25 C, 37 C, 40 C, and 45 C to determine the negatively stained for TEM analysis. The characteristic intracellular optimal growth temperature. granules are indicated by arrows.

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stably maintained until 72 h of cultivation. However, the phylogenetically related Gammaproteobacteria. Moreover, agarase activity produced by G7T started to sharply increase many intracellular granules were observed in the electron from 6 h, reached its maximum level (0.425 unit/ml) at 12 h, micrographs of the negatively-stained cells (Fig. 2). and then decreased rapidly after 24 h of cultivation (Fig. 1B). Similarly, the agarase production in Alteromonas macleodii Phylogenetic Analysis of the G7T Strain subsp. GNUM08120 was recently reported to reach its The 16S rRNA gene sequence (1,436 bp) of strain G7T was maximum level at 12 h and then sharply decreased to basal deposited in GenBank (Accession No. JF965427). A BLASTN level at 18 h of cultivation [5], which may be resulted from similarity search [2] of the G7T 16S rRNA gene revealed low concentration of carbon source (agar) in the medium. that it shares 95.5%, 90.6%, and 90.0% similarity with C. Agar is a heterogeneous polysaccharide, mainly composed agarivorans YM01T (1,379 bp), Algicola sagamiensis B-10-31T of 3,6-anhydro-L-galactoses and D-galactoses alternately (1,435 bp), and Bowmanella pacifica W3-3AT (1,436 bp), linked by α-(1,3) and β-(1,4) linkages. Therefore, agarases respectively. The 16S rRNA gene sequence of strain G7T are classified according to their cleavage pattern into two shares less than 90% similarity with strains of the genera types, α-agarase and β-agarase [6]. Although hundreds of Vibrio, Cowellia, and Glaciecola. An NJ phylogenetic tree agar hydrolyzing bacteria have been reported, most of based on the 16S rRNA gene sequence revealed that strain them were turned out to produce β-agarase but not α- G7T formed a distinct lineage within the Gammaproteobacteria agarase. According to the genomic sequencing data, it was of the genera Catenovulum, Algicola, Bowmanella, and Alteromonas also expected that strain G7T had the genes encoding only (Fig. 3). This tree topology was identical to that of phylogenetic putative β-agarase, which needs more investigation [24]. trees generated using the MP and ML algorithms. Based on TEM observations with negative staining Because the 16S rRNA gene sequence similarity was on (Fig. 2), strain G7T is non-flagellated. Its absence of motility the boundary of the criterion (less than 95%) for discriminating is a distinctive characteristic that distinguishes it from other a new genus, DNA-DNA hybridization studies were

Fig. 3. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain G7T and other representative type strains of the genera Catenovulum, Algicola, Vibrio, Alteromonas, Glaciecola, Bowmanella, and . Bootstrap values >50% based on 1,000 replicates are shown at the branch points. Bar, 0.01% estimated sequence divergence.

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Fig. 4. Phylogenomic tree of strain G7T and the sequenced strains of related taxa. The tree was constructed using the neighbor-joining method in MEGA5 [32] and the concatenated amino acid sequences of 26 broadly conserved genes [9]. The 27 proteins shown are arginyl-tRNA synthetase, GTP-dependent nucleic acid-binding protein EngD, isoleucyl-tRNA synthetase, leucyl-tRNA synthetase, LSU ribosomal proteins L5p, L6p, L13p, L15p, L16p, L18p, L22p, L33p, methionyl-tRNA synthetase, phenylalanyl-tRNA synthetase alpha chain, seryl-tRNA synthetase, SSU ribosomal proteins S2p, S3p, S4p, S5p, S8p, S9p, S11p, S13p, S15p, S17p, and valyl-tRNA synthetase. The evolutionary distances were computed using the Poisson correction method, and bootstrap values (1,000 replicates) of more than 50% are shown at the branch points. Chromohalobacter salexigens DSM 3043T was used as an outgroup. performed with strain G7T and C. agarivorans YM01T. The Catenovulum as a sister taxon of G7T, although the branch resulting DNA-DNA hybridization value was 19.63%, which lengths of the two appear to be rather extended compared is low enough to classify strain G7T as a new genus. Based with other branches in the same genus (Fig. 4). When the on the 16S rRNA gene sequence similarity, the phylogenetic average nucleotide identity values based on BLAST (ANIb; tree, and the DNA-DNA hybridization result, strain G7T [29]) were examined, the genomes of G7T and C. agarivorans appeared to belong either to a novel genus within the class YM01T had an average ANIb value of 70.00 ± 0.26% (Table 1). Gammaproteobacteria or to the genus Catenovulum. In contrast, the ANIb values between sequenced genomes As the genome sequence of strain G7T has been recently in the order that are in the same genus determined (GenBank Accession No. AMRX00000000; [24]), were 70.36 ± 0.06% for loihiensis L2TRT and its sequence and those of closely related taxa with available Idiomarina baltica OS145T, 72.00 ± 2.25% for 10 type strains genome sequences were analyzed. The phylogenomic tree of of spp., and 78.68 ± 8.44% for four type strains of G7T and sequenced type strains in the order Alteromonadales spp. This genomic sequence analysis also suggests based on 26 broadly conserved protein sequences also placed that strain G7T is distinct from the genus Catenovulum.

November 2013 ⎪ Vol. 23⎪ No. 11 1514 Chi et al. 7 9 17 .5 .97 6.44 64.93 Glaciecola SB2B; 11, Shewanella 1 65.31 - 65.42 .97 --- 26 27 63.47 65.58 62.98 65.11 63.92 65.95 Marinobacter algicola WP3; 17, 96.62 ATCC 27126; 4, --- 74.19 74.09 HP15; 23, --- 75.5 75.79 75.78 Shewanella Shewanella piezotolerans 22 23 24 25 --- DSM 9799; 10, 10, 9799; DSM Alteromonas macleodii 65.5 75.32 65.48 66.13 75.79 74.37 Marinobacter adhaerens YM01; 3, 3, YM01; 20 21 66.07 66.266.17 66.82 65.34 66.72 65.25 67.1766.31 65.31 67.16 65.13 66.01 65.5 65.26 67.97 65.76 65.7 67.34 65.94 66.74 65.49 66.4266.16 65.48 66.38 65.56 65.49 66.51 65.44 67.21 66.4866.22 65.52 66.85 66.03 65.77 65.87 67.74 65.61 65.665.97 66.98 65.28 65.4 67.18 65.1166.39 65.58 64.99 65.72 65.65 65.21 65.56 67.54 65.3766.56 65.66 66.92 67.27 66.14 65.77 66.87 66.41 65.83 66.38 67.27 66.34 67.2 66.49 67.47 67.63 ATCC 700345; 16, T7901; 22, balearica --- 66.28 65.69 65.55 65.58 65.73 65.5 67.4 67.06 K-12 MG1655. 74.41 37; 9, 9, 37; --- 75.21 75.29 Shewanella pealeana Catenovulum Catenovulum agarivorans --- Escherichia coli Escherichia 72.51 71.83 72.26 Teredinibacter turnerae G7; 2, 2, G7; --- 75.65 72.78 72.4 73.32 MR-1; 15, 15, MR-1; Psychromonas ingrahamii Psychromonas 2-40; 21, 21, 2-40; --- 71.44 71.05 71.13 70.81 71 ArB-0140; and 27, 27, and ArB-0140; --- 71.03 71.8 70.87 72.45 72.12 71.77 OS145; 8, Gayadomonas joobiniege --- 71.9 71.4 82.19 75.74 72.88 72.43 73.08 Moritella dasanensis Moritella --- 71.19 70.69 72.4 71.22 70.77 70.65 70.68 70.75 Idiomarina baltica Idiomarina PV-4; 14, --- 70.2 69.5 71.57 70.76 69.53 68.95 69.75 69.57 69.15 L2TR; 7, 7, L2TR; 10 11 12 13 14 15 16 17 18 19 70.9 72.99 71.55 71.09 69.2 70.83 73.3 71.99 71.14 73.41 72.53 75.26 74.15 ATCC 49840; 26, 26, 49840; ATCC 70.26 69.69 71.24 71.45 70.69 71.86 69.59 71.29 82.2668.87 71.9 70.6 71.43 69.72 75.7 70.79 70.83 72.9769.85 70.94 72.64 71.04 75.5 71.1 72.61 72.43 72.81 71.09 72.58 72.65 72.2 75.28 ATCC 51908; 20, Shewanella loihica 89 66.02 65.76 65.78 65.79 65.87 65.61 65.85 65.91 65.84 65.85 65.83 65.66 65.35 65.1 65.27 65.31 65.87 65.24 65.85 66.29 HAW-EB4; 13, 13, HAW-EB4; --- 65.07 65.32 65.5 65.72 65.6 65.6 65.96 65.79 65.79 65.43 65.49 65.53 64.83 64.69 64.45 64.77 65.24 64.5 65.69 65.53 70.4 67 70.32 KCTC 22429; 6, 22429; KCTC DSS12; 19, Marinobacter hydrocarbonoclasticus Shewanella halifaxensis VT8; 25, 25, VT8; Shewanella Shewanella violacea jeotgali 345 OS217; 12, 66.27 66.59 66.09 66.55 66.03 66.73 65.18 65.49 66.42 66.39 65.81 66.3 66.43 66.3 66.17 66.08 66.44 65.5 65.05 64.41 64.3 64.71 64.57 66.61 65.87 --- 65.81 65.74 65.19 65.57 65.46 66.12 63.7 64.47 65.61 65.72 64.4 65.69 65.73 65.6 65.16 65.03 65.14 64.36 63.62 62.77 62.74 62.86 62.86 66.7 64.77 70.18 FR1064; 5, 5, FR1064; Marinobacter aquaeolei Average nucleotide identity values of G7 and closely related sequenced strains in the family Alteromonadaceae. Alteromonadaceae. family the in strains sequenced related closely and of G7 values identity nucleotide Average HAW-EB3; 18, 12 --- 65.8 64.97 66.61 65.47 67.17 66.39 65.69 65.93 68.93 --- 66.11 65.58 66.51 66 66.55 --- 66.74 66.35 66.8966.75 66.18 66.09 66.65 66.84 66.4566.01 66.16 66.04 65.06 66.59 66.98 66.63 66.81 66.68 67.22 65.93 65.6 66.46 67.18 66.89 66.88 66.63 66.58 66.22 66.38 65.87 66.99 65.82 67.19 66.46 65.8566.68 66.14 66.05 68.87 66.39 66.4 66.41 67.02 66.89 66.72 66.7 66.15 65.69 67.07 66.08 67.04 66.43 66.95 66.27 66.24 66.27 66.52 65.4 66.05 66.6966.98 67.16 67.01 66.14 65.87 66.57 66.23 67.05 66.61 66.78 66.87 66.21 67.13 66.23 66.36 67.46 66.5 67.09 65.92 67.0463.63 66.72 62.57 64.663.03 62.65 63.37 64.16 65.0763.82 63.27 64.95 64.71 64.34 63 64.59 63.4364.06 64.44 66.53 63.01 64.89 63.06 65.61 64.85 63.57 66.12 64.17 64.01 65.67 65.66 64.21 65.53 65.7 63.92 65.35 65.01 63.86 65.43 64.1 63.28 65.26 64.8 64.06 66.8 63.97 64.04 63.63 63.89 66.32 66.85 63.39 64.22 64 66.26 63.97 64.59 64.57 63.84 65.78 64.29 64.61 63.72 64.63 63.99 65.75 64.64 64.35 64.88 65.1 64.13 64.7 64.66 64.58 64.28 64.23 64.71 65.31 64.56 64.53 65.2 66.38 75.78 74.14 96.73 --- 63.85 65. 8 679 65.01 66.23 66.64 64 66.62 66.26 66.05 67.13 64.72 65.97 66.98 66.02 --- 65.57 65.08 65.76 65.77 --- 66.87 67.04 68.87 66.09 66.42 66.79 66.34 66.86 68.65 66.98 66.59 67.03 66.17 66.9 65.87 66.57 66.98 66.45 66.24 66.19 65.99 65.13 64.83 65.67 64.98 66.97 64.93 66.71 64.96 67.26 68.37 65 67.1 65.1 68. 1 2 69.81 3 66.12 66.45 --- 68.12 65.96 66.56 66.12 65.94 65.65 65.87 66 66.35 66.1 66.17 66.46 66.37 66.13 65.95 65.94 65.62 64.91 64.94 64.89 64.96 64.96 66.3 66.0 4 66.33 65.395 67.93 65.62 65.28 --- 65.76 65.21 65.03 65.89 --- 65.43 65.34 66.19 64.49 65.58 64.6 65.43 66.54 65.17 65.31 66.5 64.95 65.72 65.38 65.75 65.34 66.35 65.39 66.2 65.1 65.62 64.98 65.45 65.19 65.47 64.78 65.59 63.92 65.42 63.55 65.15 63.53 65.13 63.85 65.17 63.78 65.08 65.93 65.64 65.6 65.41 6 7 65.81 65.7 65.91 65.41 65.92 26 65.87 66.3327 65.99 65.53 65.47 64.57 65.03 66.49 65.56 65.79 65.31 67.06 67.42 66.74 64.16 65.87 64.89 65.64 66.16 66.55 68.3 65.31 67.88 66.18 66.62 66.54 66.47 66.44 67.48 66.1 67.03 66.54 66.3 66.21 66.51 66.43 66.63 64.74 63.58 66.3 63.39 65.53 63.12 65.82 63.55 66.33 63.53 66.19 66.51 -- 66.66 65 10 11 12 13 14 15 16 17 18 19 22 23 24 25 20 64.95 64.3721 64.9 64.2 64.43 63.58 64.75 64.65 64.99 63.95 64.31 64.56 64.46 65.03 64.36 64.11 64.58 64.46 64.9 65.33 64.72 64.52 64.08 64.78 64.27 64.9 64.57 64.69 64.37 64.12 64.4 63.96 64.39 64.24 64.36 63.93 64.13 63.95 67.44 ------67.45 65.12 65.84 65.04 65.65 65.24 66.07 65.24 66.08 64.9 64. 64 sediminis Table 1. Jspecies [29] was used to calculate the ANIb values and alignment percentages. The strains are 1, Shewanella denitrificans DG893; 24, nitroreducens

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T Physiological and Biochemical Analyses of Strain G7 Table 2. Phenotypic characteristics of strain G7T and Catenovulum Strain G7T grows at 20–42oC, but does not grow at 4oC, agarivorans YM01T. o o 10 C, or 50 C. It grows well between pH 6 and 9, but does Characteristics Strain G7T C. agarovarans YM01T not grow at pH 4, 5, or 10. Growth and agarase activity Source Sea water, Gaya Sea water, were observed in ASW-YP liquid medium containing 0.5– Island, Korea Qingdao, China 5% (w/v) NaCl; however, no growth was observed in G+C content (mol%) 41.12 40.04 T medium containing 0% or 6% NaCl (w/v). Strain G7 was Flagella - + highly susceptible to chloramphenicol, but resistant to all Colony color White Yellow other antibiotics tested. Growth at Strain G7T is differentiated from its nearest phylogenetic 4oC-- neighbor, C. agarivorans YM01T, by several phenotypic 42oC++ characteristics, which are shown in Table 2. In particular, pH 5 - - we confirmed that the major isoprenoid quinone of strain pH 9 + + G7T is ubiquinone-8 (Q-8), whereas that of C. agarivorans YM01T is menaquinone-7. Other quinones were not detected pH12 - - in the strains G7T and YM01T, coinciding with the previous 0.5% NaCl + + report. Q-8 is a predominant quinone in other relevant species 5% NaCl + - of the genera Algicola [22], Vibrio [7], Bowmanella [17, 25], 8% NaCl - - Alteromonas [15], Pseudoalteromonas [22], Glaciecola [4, 36], Carbon utilization: and Aestuariibacter [34, 38], but several other ubiquinones D-Glucose + + have also been detected in species of B. denitrificans (11.1% D-Mannitol + - Q-9 and 7.4% Q-10), A. hispanica (3.5% Q-7,) and A. simiduii D-Melibiose - + (4.3% Q-4 and Q-6) with a relative small amount [8, 17, 26]. Glycerol + - T The isoprenoid quinone analysis classifies strain G7 as a D-Lactose + - T novel genus distinct from C. agarivorans YM01 . The DNA D-Trehalose + - T G+C content of strain G7 was calculated from its genomic Glycogen + - sequence as 41.12 mol%. This value is consistent with the D-Turanose + -

DNA G+C content of the reference Gammaproteobacteria D-Galactose w + strains. Strain G7T was resistant to ampicillin, kanamycin, and L-Arabinose - + T neomycin, but C. agarivorans YM01 was susceptible to them. N-Acetyl-glucosamine - + The cellular fatty acid compositions of strain G7T and Inositol + - C. agarivorans YM01T grown on MB plates for 96 h are Enzyme activity: shown in Table 3. The predominant fatty acids of strain G7T Esterase (C4) W + are Iso-C (41.47%), Anteiso-C (22.99%), and summed 15:0 15:0 Esterase (C8) - + feature 3 comprising C16:1ω7c/iso-C15:0 2-OH (8.85%), whereas T Leucine arylamidase V + those of YM01 were summed feature 3 (37.48%), C16:0 Valine arylamidase - + (18.77%), and C15:0 (10.63%). The two major components of T Cystine arylamidase - W strain G7 , Iso-C15:0 and Anteiso-C15:0, were not detected in C. agarivorans YM01T. In addition, other cellular fatty acids Trypsin - + were present at significantly different levels in strain G7T α-Chymotrypsin - W and C. agarivorans YM01T. To conclude, phylogenetic, β-Galactosidase + - chemotaxonomic, and physiological analyses showed that β-Glucosidase - + strain G7T does not belong to any previously described Nitrate to nitrite + + genus. Therefore, strain G7T is classified as a novel genus Isoprenoid quinone Ubiquinone-8 Menaquinone-7 and species, for which the name Gayadomonas joobiniege Antibiotics susceptibility gen. nov. sp. nov. is proposed. Ampicillin - + Kanamycin - + Description of Gayadomonas gen. nov. Neomycin - + Gayadomonas (Ga.ya.do.mo’nas. N.L. n. Gayado, an island Symbol : +, positive; -, negative; W, weak positive; V, very weak positive

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Table 3. Cellular fatty acid composition (1%) of strain G7T and Colonies are 1.5 mm in size when grown on ASW-YP for Catenovulum agarivorans YM01T. 48 h at 40oC. Colonies are opaque and white when grown Fatty acid Strain G7T C. agarovarans YM01T on marine agar (Difco 2216) and ASW-YP agar for 48 h at 40oC, with smooth-rounded surfaces and entire margins. C10:0 3-OH 1.36 5.90 Cells grow in culture media containing 0.5–5% NaCl (w/v), C12:0 0.50 1.94 with optimum growth in 3% NaCl (w/v). Cells grow well C 0.16 2.08 13:0 at 20oC and 42oC, with optimum growth at 35–40oC, but not Iso-C 1.25 ND 14:0 at 4oC or 50oC. Cells grow well at pH 6.0 and 9.0, with C 0.68 2.78 14:0 optimum growth at pH 7.0. Agar is hydrolyzed. Glycerol, Iso-C15:0 41.47 ND D-ribose, D-glucose, D-fructose, inositol, gentiobiose, D- Anteiso-C 22.99 ND 15:0 tagatose, gluconate, D-xylose, methyl-β D-xylopyranoside, C 1.17 10.63 15:0 D-galactose, D-mannose, D-mannitol, amygdalin, salicin, D- C ω6c 0.05 1.19 15:1 cellobiose, D-maltose, D-lactose, D-saccharose, D-trehalose, C ω7c alcohol 1.80 ND 16:1 D-raffinose, starch, glycogen, and D-turanose are utilized as C ω11c 1.66 ND 16:1 a sole carbon source. Erythritol, D-melibiose, D-arabinose, C 4.59 18.77 16:0 L-xylose, D-adonitol, L-sorbose, L-rhamnose, dulcitol, D- Iso-C ω10c 2.69 ND 17:1 sorbitol, methyl-α D-mannopyranoside, methyl-α D-

Iso-C17:0 1.69 ND glucopyranoside, arbutin, esculin ferric citrate, inulin, D-

C17:0 0.86 2.33 melezitose, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitol,

C18:1 ω9c 0.12 2.01 L-arabitol, 2-ketogluconate, N-acetylglucosamine, L-arabinose,

C18:1 ω7c 0.96 4.68 and 5-ketogluconate are not utilized as a sole carbon source.

C18:0 0.15 2.62 Acidification from D-glucose, D-fructose, D-mannose, D- Summed featurea: maltose, D-trehalose, D-mannitol, D-xylose, and, D-saccharose 3 8.85 37.48 was observed. Acidification from D-lactose, xylitol, D- 4 1.90 ND melibiose, D-raffinose, and methyl-α D-glucopyranoside 5 0.13 2.07 was not observed. Reduction of nitrates to nitrites and Unkown ECL 11.799 0.56 2.69 indole production are positive and weakly positive, ND, not detected. respectively; however, acetyl-methyl-carbinol production a is negative. Arginine dihydrolase, urease, α-galactosidase, β- Sum 3, C16:1 ω7c/iso-C15:0 2-OH; 4, C17:1 Iso I/Anteiso B; 5, C18:2 ω6c, 9c/Anteiso-

C18:0. galactosidase, gelatinase, alkaline phosphatase, esterase (C4), leucine arylamidase, acid phosphatase, naphthol-AS-BI- phosphohydrolase, lipase (C14), and α-glucosidase are located in the southern region of the Republic of Korea; L. produced, but esterase (C8), cystine arylamidase, valine fem. n. monas, a unit, monad; N.L. fem. n. Gayadomonas, a arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, β- monad from Gayado). Gram-negative, aerobic, mesophilic, glucosidase, N-acetyl-β-glucosaminase, α-mannosidase, rod-shaped cells belonging to the class Gammaproteobacteria. and α-fucosidase are not. Susceptibility to chloramphenicol Intracellular granules are observed. NaCl is an absolute was observed, but not to ampicillin, neomycin, kanamycin, requirement for growth. Nitrate reduction, indole production, apramycin, or thiostrepton. Ubiquinone-8 is the major glucose utilization, and acidification are positive. Cells grow respiratory quinone. The major fatty acids are Iso-C15:0 o o o well at 20–42 C but not at 4 C or 50 C. The major isoprenoid (41.47%), Anteiso-C15:0 (22.99%), and summed feature 3 quinone is Q-8. Predominant cellular fatty acids are Iso-C15:0 comprising C16:1ω7c/iso-C15:0 2-OH (8.85%). T T and Anteiso-C15:0. The type species is Gayadomonas joobiniege. The type strain, G7 (ATCC BAA-2321 = DSM25250 = KCTC23721T) was isolated from the seatwater of Gaya Island, Description of Gayadomonas joobiniege sp. nov. Republic of Korea. The DNA G+C content is 41.12 mol%. Gayadomonas joobiniege (joo.bi’ni.e.ge. N.L. fem. n. joobiniege of Joobin, the given name of the sample collector) GenBank Accession Number Cells are non-flagellated and rod-shaped (1.0–1.2 µm The GenBank/EMBL/DDBJ accession number for the wide and 3.5–5.5 µm long) when grown on solid media. 16S rRNA gene sequence of strain G7T is JF965427.

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