Introduction

Marine microorganisms have received a great deal of attention in various efforts to utilize the abundant biological resources in the sea. One of these resources is agar, which is a major component of red algae [6]. Agar is widely used as a dietary ingredient in food and as a gelling agent in solid culture media for microbial growth. Recently, agar oligosaccharides have also been reported to have antioxidant activity, therapeutic activity in inflammatory disease, and antitumor activity, etc. [3,11,19], which may broaden its application in the food, cosmetic, and pharmaceutical industries as well as biorefinement or biofuel industries.

For an efficient hydrolysis of agar, research seeking a good agarase has been under intense spotlight. A number of microorganisms with agar-hydrolyzing activity, such as Pseudoalteromonas marinoglutinosa [30], Alteromonas atlantica [1], Glaciecola 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 marinebacterium (designated as strain G7T), which can grow on minimal medium supplemented with agar as the sole carbon source at higher temperature (above 40℃). 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).

Isolation of Agarase-Producing Microorganisms

Coastal seawater of Gaya Island, Republic of Korea, was collected to isolate agar-degrading bacteria. The collected sample was serially diluted from 10-1 to 10-5, and 200 μl of each dilution was smeared on an artificial seawater (ASW) agar plate [20] containing 1.0% yeast extract (w/v) and 0.3% bacto peptone (w/v) (ASW-YP), and was incubated aerobically at 40℃ for 24 h. The ASW contained 6.1 g Tris base, 12.3 g MgSO4, 0.74 g KCl, 0.13 g (NH4)2HPO4, 17.5 g NaCl, and 0.14 g CaCl2 d issol ved in 1 L o f distilled water. A total of 1,136 colonies were collected and transferred to fresh ASW-YP plates and incubated at 40℃ for 24 h. The plate was stained with Lugol’s iodine solution (0.05 M iodine in 0.12 M KI) to detect agarase activity (Fig. 1A). Colonies with high agarase activity were isolated from the replica plate and transferred to a fresh ASW-YP plate. The bacterial colonies were streaked five times to obtain a single colony for pure culture. One marine bacterium with agar-hydrolyzing activity was selected and designated as strain G7T in this study. After incubation in ASW-YP broth at 40℃ for 1 day, the cul ture was supplemented with 10% glycerol (w/v) and stored at -80℃ as the stock culture.

Production of Agarase by Strain G7T

Cell growth and agarase activity of strain G7T were observed by incubating it in ASW-YP liquid medium containing 0.1% agar (w/v) at 40℃ for 72 h (Fig. 1B). Then, 1 ml of culture broth was sampled at regular time intervals, and the optical density (OD) was measured at 600 nm (OD600) to plot the growth curve. The sample was centrifuged at 14,000 rpm for 10 min and its supernatant was collected to measure agarase activity. A substrate solution containing 0.2% agarose in 10 mM Tris-HCl (pH8.0) was used for the agarase reaction. Agarase activity was measured by the previously described method [27] using 3,5-dinitrosalicylic acid (DNS). One unit (U) of agarase was defined as the amount of enzyme that produced 1 μmol galactose per minute at 40℃. Galactose was used as a reference reducing sugar for preparing the standard curve.

Fig. 1.Agarase production by strain G7T. (A) Detection of agarolytic activity of strain G7T on agar plate. The strain was cultured on an ASW-YP agar plate at 40℃ for 2 days (left), and Lugol’s iodine solution was overlaid to detect reducing sugars and degraded product from agar by agarase (right). (B) Cell growth and agarase production of strain G7T in ASW-YP broth depending on cultivation time. The agarolytic activity was estimated by the colorimetric DNS method to measure the reducing sugar that resulted from hydrolysis of agar as the substrate. Each value is an average of three parallel replicates. ●-● , Cell growth; ■-■, agarase activity.

16S rRNA Sequencing and Construction of Phylogenetic Tree

The bacterial strain was cultured in ASW-YP liquid medium for 3 days, and genomic DNA was extracted with a genomic DNA extraction kit (Promega Co., USA). The 16S rRNA gene was amplified by PCR u sing u niversal bacterial primers (27F, 5’-AGAGTTTGATCCTGGCTCAG-3’; and 1492R, 5’-TACCTTGTTACGACTT-3’), and nucleotide sequencing was performed using an Applied Biosystems 3730xl DNA Analyzer. Analysis of the 16S rRNA gene sequence revealed that strain G7T belongs to the class Gammaproteobacteria. The 16S rRNA gene sequences of type strains related to strain G7T were collected from the EzTaxon server (http://www.eztaxon.org; [9]). The 16S rRNA sequences were aligned using ClustalW software [35], and the 5’ and 3’ gaps were edited using the BioEdit program [13]. Neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) methods from the PHYLIP suite program [12] were used to construct the phylogenetic tree. The bootstrap value was calculated using data restructured nearly 1,000 times and marked into the branching point. The evolutionary distance matrix was estimated according to Kimura’s 2-parameter model [21].

DNA-DNA Hybridization

Genomic DNAs from strain G7T and the phylogenetically closest type strain C. agarivorans YM01 were used for DNA-DNA hybridization. Escherichia coli KCCM12119T was used as a negative control. DNA probe preparation and the hybridization reaction were performed with the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Germany) according to the manufacturer’s instruction. The resulting hybridization signals were measured using the Quantity One Program (Bio-Rad, USA). The signal from strain G7T was set at 100%.

Phenotypic and Biochemical Characteristics

Bacterial cells were grown on ASW-YP agar plates for 2 days and their morphology, size, and motility were examined by phasecontrast 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 28℃ 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 incubated at 40℃ for 3 days to investigate the effect of initial pH on growth. The isolate was inoculated on ASW-YP agar plates and incubated at 4℃, 15℃, 25℃, 37℃, 40℃, and 45℃ to determine the optimal growth temperature.

To determine antibiotic susceptibility, strain G7T and C. agarivorans YM01 were smeared on ASW-YP agar plates and incubated at 40℃ for 1 h. Paper discs containing 30 μl of thiostrepton (100 μg/ml), kanamycin (100 μg/ml), neomycin (100 μg/ml), ampicillin (100 μg/ml), apramycin (100 μg/ml), and chloramphenicol (100 μg/ml) were placed on the smear plate. The plates were incubated at 40℃ for 24 h and the zone of inhibition surrounding each antibiotic disc was measured.

Chemotaxonomic Characteristics

The major respiratory quinones of strain G7T and C. agarivoransYM01 were analyzed by reverse-phase high-performance liquid chromatography (HPLC) after growth on Marine Broth (MB; Difco) plates [23]. Cellular fatty acid methyl ester (FAME) mixtures were prepared from G7T and YM01 cells grown on MB (Difco) plates for 4 days by methyl esterification [28] and analyzed by gas chromatography using the Microbial Identification software package [32].

Results and Discussion

Phenotypic Characteristics of the Agar-Hydrolyzing Isolate, G7T

Strain G7T is a gram-negative, rod-shaped bacterium. It forms non-pigmented colonies that are circular and smooth and produces agarases (Fig. 1A). Strain G7T produces colonies that are approximately 1.5 mm in diameter after incubation on an ASW-YP agar plate at 40℃ for 48 h. The growth of strain G7T in ASW-YP broth containing 0.1% agar as the sole carbon source started to increase from 3 h, reached its maximum level (OD600=0.85) at 36 h, and was stably maintained until 72 h of cultivation. However, the agarase activity produced by G7T started to sharply increase from 6 h, reached its maximum level (0.425 unit/ml) at 12 h, and then decreased rapidly after 24 h of cultivation (Fig. 1B). Similarly, the agarase production in Alteromonas macleodii subsp. GNUM08120 was recently reported to reach its maximum level at 12 h and then sharply decreased to basal level at 18 h of cultivation [5], which may be resulted from low concentration of carbon source (agar) in the medium.

Fig. 2.Transmission electron microscopy (TEM) of strain G7T. The strain was grown on an ASW-YP plate at 40℃ for 2 days and was negatively stained for TEM analysis. The characteristic intracellular granules are indicated by arrows.

Agar is a heterogeneous polysaccharide, mainly composed of 3,6-anhydro-L-galactoses and D-galactoses alternately linked by α-(1,3) and β-(1,4) linkages. Therefore, agarases are classified according to their cleavage pattern into two types, α-agarase and β-agarase [6]. Although hundreds of agar hydrolyzing bacteria have been reported, most of them were turned out to produce β-agarase but not α- agarase. According to the genomic sequencing data, it was also expected that strain G7T had the genes encoding only putative β-agarase, which needs more investigation [24].

Based on TEM observations with negative staining (Fig. 2), strain G7T is non-flagellated. Its absence of motility is a distinctive characteristic that distinguishes it from other phylogenetically related Gammaproteobacteria. Moreover, many intracellular granules were observed in the electron micrographs of the negatively-stained cells (Fig. 2).

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 Aestuariibacter. Bootstrap values >50% based on 1,000 replicates are shown at the branch points. Bar, 0.01% estimated sequence divergence.

Phylogenetic Analysis of the G7T Strain

The 16S rRNA gene sequence (1,436 bp) of strain G7T was deposited in GenBank (AccessionNo.JF965427). A BLASTN similarity search [2] of the G7T 16S rRNA gene revealed that it shares 95.5%, 90.6%, and 90.0% similarity with C. agarivorans YM01T (1,379 bp), Algicola sagamiensis B-10-31T (1,435 bp), and Bowmanella pacifica W3-3AT (1,436 bp), respectively. The 16S rRNA gene sequence of strain G7T shares less than 90% similarity with strains of the genera Vibrio, Cowellia, and Glaciecola. An NJ phylogenetic tree based on the 16S rRNA gene sequence revealed that strain G7T formed a distinct lineage within the Gammaproteobacteria of the genera Catenovulum, Algicola, Bowmanella, and Alteromonas(Fig. 3). This tree topology was identical to that of phylogenetic trees generated using the MP and ML algorithms.

Because the 16S rRNA gene sequence similarity was on the boundary of the criterion (less than 95%) for discriminating a new genus, DNA-DNA hybridization studies were performed with strain G7T and C. agarivorans YM01T. The resulting DNA-DNA hybridization value was 19.63%, which is low enough to classify strain G7T as a new genus. Based on the 16S rRNA gene sequence similarity, the phylogenetic tree, and the DNA-DNA hybridization result, strain G7T appeared to belong either to a novel genus within the class Gammaproteobacteria or to the genus Catenovulum.

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.

As the genome sequence of strain G7T has been recently determined (GenBank Accession No. AMRX00000000; [24]), its sequence and those of closely related taxa with available genome sequences were analyzed. The phylogenomic tree of G7T and sequenced type strains in the order Alteromonadales based on 26 broadly conserved protein sequences also placed Catenovulum as a sister taxon of G7T, although the branch lengths of the two appear to be rather extended compared with other branches in the same genus (Fig. 4). When the average nucleotide identity values based on BLAST (ANIb; [29]) were examined, the genomes of G7T and C. agarivorans YM01T had an average ANIb value of 70.00 ± 0.26% (Table 1). In contrast, the ANIb values between sequenced genomes in the order Alteromonadales that are in the same genus were 70.36 ± 0.06% for Idiomarina loihiensis L2TRT and Idiomarina baltica OS145T, 72.00 ± 2.25% for 10 type strains of Shewanella spp., and 78.68 ± 8.44% for four type strains of Marinobacter spp. This genomic sequence analysis also suggests that strain G7T is distinct from the genus Catenovulum.

Table 1.Jspecies [29] was used to calculate the ANIb values and alignment percentages. The strains are 1,Gayadomonas joobiniege G7; 2, Catenovulum agarivorans YM01; 3, Alteromonas macleodii ATCC 27126; 4, Glaciecola nitroreducens FR1064; 5, Alishewanella jeotgali KCTC 22429; 6, Idiomarina loihiensis L2TR; 7, Idiomarina baltica OS145; 8, Psychromonas ingrahamii 37; 9, Ferrimonas balearica DSM 9799; 10, Shewanella amazonensis SB2B; 11, Shewanella denitrificans OS217; 12, Shewanella halifaxensis HAW-EB4; 13, Shewanella loihica PV-4; 14, Shewanella oneidensis MR-1; 15, Shewanella pealeana ATCC 700345; 16, Shewanella piezotolerans WP3; 17, Shewanella sediminis HAW-EB3; 18, Shewanella violacea DSS12; 19, Shewanella woodyi ATCC 51908; 20, Saccharophagus degradans 2-40; 21, Teredinibacter turnerae T7901; 22, Marinobacter adhaerens HP15; 23, Marinobacter algicola DG893; 24, Marinobacter aquaeolei VT8; 25, Marinobacter hydrocarbonoclasticus ATCC 49840; 26, Moritella dasanensis ArB-0140; and 27, Escherichia coli K-12 MG1655.

Physiological and Biochemical Analyses of Strain G7T

Strain G7T grows at 20–42℃, but does not grow at 4℃, 10℃, or 50℃. It grows well between pH 6 and 9, but does not grow at pH 4, 5, or 10. Growth and agarase activity were observed in ASW-YP liquid medium containing 0.5–5% (w/v) NaCl; however, no growth was observed in medium containing 0% or 6% NaCl (w/v). Strain G7T was highly susceptible to chloramphenicol, but resistant to all other antibiotics tested.

Strain G7T is differentiated from its nearest phylogenetic neighbor, C. agarivorans YM01T , by several phenotypic characteristics, which are shown in Table 2. In particular, we confirmed that the major isoprenoid quinone of strain G7T is ubiquinone-8 (Q-8), whereas that of C. agarivorans YM01T is menaquinone-7. Other quinones were not detected in the strains G7T and YM01T, coinciding with the previous report. Q-8 is a predominant quinone in other relevant species of the genera Algicola [22], Vibrio [7], Bowmanella [17,25], Alteromonas [15], Pseudoalteromonas [22], Glaciecola [4,36], and Aestuariibacter [34,38], but several other ubiquinones have also been detected in species of B. denitrificans (11.1% Q-9 and 7.4% Q-10), A. hispanica (3.5% Q-7,) and A. simiduii (4.3% Q-4 and Q-6) with a relative small amount [8,17,26]. The isoprenoid quinone analysis classifies strain G7T as a novel genus distinct from C. agarivorans YM01T. The DNA G+C content of strain G7T was calculated from its genomic sequence as 41.12 mol%. This value is consistent with the DNA G+C content of the reference Gammaproteobacteria strains. Strain G7T was resistant to ampicillin, kanamycin, and neomycin, but C. agarivorans YM01T was susceptible to them.

The cellular fatty acid compositions of strain G7T and C. agarivorans YM01T grown on MB plates for 96 h are shown in Table 3. The predominant fatty acids of strain G7T are Iso-C15:0 (41.47%), Anteiso-C15:0 (22.99%), and summed feature 3 comprising C16:1ω7c/iso-C15:0 2-OH (8.85%), whereas those of YM01T were summed feature 3 (37.48%), C16:0(18.77%), and C15:0 (10.63%). The two major components of strain G7T, Iso-C15:0 and Anteiso-C15:0, were not detected in C. agarivorans YM01T. In addition, other cellular fatty acids were present at significantly different levels in strain G7T and C. agarivorans YM01T . To conclude, phylogenetic, chemotaxonomic, and physiological analyses showed that strain G7T does not belong to any previously described genus. Therefore, strain G7T is classified as a novel genus and species, for which the name Gayadomonas joobiniege gen. nov. sp. nov. is proposed.

Description of Gayadomonas gen. nov.

Gayadomonas (Ga.ya.do.mo’nas. N.L. n. Gayado, an island located in the southern region of the Republic of Korea; L. fem. n. monas, a unit, monad; N.L. fem. n. Gayadomonas, a monad from Gayado). Gram-negative, aerobic, mesophilic, rod-shaped cells belonging to the class Gammaproteobacteria. Intracellular granules are observed. NaCl is an absolute requirement for growth. Nitrate reduction, indole production, glucose utilization, and acidification are positive. Cells grow well at 20–42℃ but not at 4℃ or 50℃. The major isoprenoid quinone is Q-8. Predominant cellular fatty acids are Iso-C15:0and Anteiso-C15:0. The type species is Gayadomonas joobiniege.

Table 2.Symbol : +, positive; -, negative; W, weak positive; V, very weak positive

Table 3.ND, not detected. aSum 3, C16:1 ω7c/iso-C15:0 2-OH; 4, C17:1 Iso I/Anteiso B; 5, C18:2 ω6c, 9c/Anteiso-C18:0.

Description of Gayadomonas joobiniege sp. nov.

Gayadomonas joobiniege (joo.bi’ni.e.ge. N.L. fem. n. joobiniege of Joobin, the given name of the sample collector)

Cells are non-flagellated and rod-shaped (1.0–1.2 μm wide and 3.5–5.5 μm long) when grown on solid media. Colonies are 1.5 mm in size when grown on ASW-YP for 48 h at 40℃. Colonies are opaque and white when grown on marine agar (Difco 2216) and ASW-YP agar for 48 h at 40℃, with smooth-rounded surfaces and entire margins. Cells grow in culture media containing 0.5–5% NaCl (w/v), with optimum growth in 3% NaCl (w/v). Cells grow well at 20℃ and 42℃, with optimum growth at 35–40℃, but not at 4℃ or 50℃. Cells grow well at pH 6.0 and 9.0, with optimum growth at pH 7.0. Agar is hydrolyzed. Glycerol, D-ribose, D-glucose, D-fructose, inositol, gentiobiose, Dtagatose, gluconate, D-xylose, methyl-β D-xylopyranoside, D-galactose, D-mannose, D-mannitol, amygdalin, salicin, Dcellobiose, D-maltose, D-lactose, D-saccharose, D-trehalose, D-raffinose, starch, glycogen, and D-turanose are utilized as a sole carbon source. Erythritol, D-melibiose, D-arabinose, L-xylose, D-adonitol, L-sorbose, L-rhamnose, dulcitol, Dsorbitol, methyl-α D-mannopyranoside, methyl-α Dglucopyranoside, arbutin, esculin ferric citrate, inulin, Dmelezitose, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, N-acetylglucosamine, L-arabinose, and 5-ketogluconate are not utilized as a sole carbon source. Acidification from D-glucose, D-fructose, D-mannose, Dmaltose, D-trehalose, D-mannitol, D-xylose, and, D-saccharose was observed. Acidification from D-lactose, xylitol, Dmelibiose, D-raffinose, and methyl-α D-glucopyranoside was not observed. Reduction of nitrates to nitrites and indole production are positive and weakly positive, respectively; however, acetyl-methyl-carbinol production is negative. Arginine dihydrolase, urease, α-galactosidase, β- galactosidase, gelatinase, alkaline phosphatase, esterase (C4), leucine arylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase, lipase (C14), and α-glucosidase are produced, but esterase (C8), cystine arylamidase, valine arylamidase, trypsin, α-chymotrypsin, β-glucuronidase, β- glucosidase, N-acetyl-β-glucosaminase, α-mannosidase, and α-fucosidase are not. Susceptibility to chloramphenicol was observed, but not to ampicillin, neomycin, kanamycin, apramycin, or thiostrepton. Ubiquinone-8 is the major respiratory quinone. The major fatty acids are Iso-C15:0(41.47%), Anteiso-C15:0 (22.99%), and summed feature 3 comprising C16:1ω7c/iso-C15:0 2-OH (8.85%).

The type strain, G7T (ATCC BAA-2321 = DSM25250T = KCTC23721T) was isolated from the seatwater of Gaya Island, Republic of Korea. The DNA G+C content is 41.12 mol%.

GenBank Accession Number

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain G7T is JF965427.