Agarivorans albus gen. nov., sp. nov., a {gamma}-proteobacterium isolated from marine animals

The objective of the present study was to shed light on the diversity of bacteria in the intestines of marine creatures, especially Mollusca. In total, 116 bacterial strains were isolated from the intestines of 21 species of marine creatures, including molluscs and protochordata, that were collected at a depth of 15 m in sea water 15 m from the shore.

Partial 16S rDNA sequencing revealed that the isolated strains belonged to the γ-Proteobacteria, α-Proteobacteria and the CytophagaFlavobacteriumBacteroides group. BLAST searches revealed that the complete 16S rDNA sequences of 17 of the 116 isolates had <94 % similarity with 16S rDNA sequences that had been deposited in the databases. Of these 17 isolates, six belonged to the Alteromonas group and six were related closely to each other, representing a novel lineage within the γ-Proteobacteria. Based on our polyphasic taxonomic study, a novel bacterium, Agarivorans albus gen. nov., sp. nov., is described here.

Sampling.
The strains used in this study were obtained from internal organs of reference marine creatures (Table 1). Marine creatures were collected by scuba-diving or by wading off the coasts of Izu-Osezaki, Shizuoka Prefecture and Izu-Ohshima Island, Japan, in 19992000.

Table 1. Isolation sources of Agarivorans albus strains and sampling sites

Isolation of bacterial strains.
The collected marine creatures were washed several times with sterile sea water. Excised gastrointestinal tracts and attached internal organs were homogenized and diluted serially to a ratio of 1 : 10 in sterile sea water. Aliquots (0·1 ml each) of the dilution were spread onto marine agar 2216 (Difco). Plates were then incubated at 23 °C for 1 week. Colonies that appeared on the plates were purified by repeated streaking and were then stored by freezing at 80 °C.

Phenotypic characterization.
Phenotypic features of the isolates, i.e. colony morphology, pigment production and agar hydrolysis, were determined by cultivating the isolates on marine agar 2216 at 23 °C. Biochemical tests were carried out by using BIOTEST (Eiken Chemical). Flagellation was observed with a model JEOL 1210 transmission electron microscope after negative staining with phosphotungstic acid.

Analyses of fatty acids and isoprenoid quinones.
For analysis of whole-cell fatty acids, cells were grown for 24 h at 23 °C on marine agar and analysed by using the GC-based Microbial Identification system (MIDI). Isoprenoid quinones were extracted with chloroform/methanol (2 : 1, v/v) and purified by TLC, using a mixture of n-hexane and diethyl ether (85 : 15, v/v) as the solvent. Ubiquinone fractions were analysed by HPLC, using the method described by Collins & Jones (1981).

Genotypic analysis.
DNA was extracted by using the method of Marmur (1961). The G+C content of the DNA was determined by HPLC (Tamaoka & Komagata, 1984). DNADNA hybridization was carried out at 45 °C with photobiotin-labelled DNA and microplates (Ezaki et al., 1989).

Phylogenetic analysis.
Template DNA for PCR was prepared by the method of Hiraishi (1992). 16S rDNA was amplified by using universal primers, as described previously (Kurahashi et al., 2002). PCR products were purified with GFX PCR DNA and a gel band purification kit (Amersham Pharmacia Biotech). Purified PCR products were sequenced directly with a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems).

The almost-complete 16S rDNA sequences determined in this work were compared with sequences in GenBank by using the BLAST algorithm (Altschul et al., 1997). Sequences were aligned with those of reference organisms derived from databases. Alignment and phylogenetic analysis were performed using CLUSTAL_W v.1.8 (Thompson et al., 1994). Phylogenetic trees were constructed, based on the K_nuc values determined by the neighbour-joining method (Saitou & Nei, 1987). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985).

16S rDNA sequences and phylogeny
The region that corresponded to the 16S rDNA could be aligned and was 1244 nt long after removal of ambiguous positions. Among the six isolates, 16S rDNA sequence similarities were 99·4100·0 %. Results of sequence alignment of these six strains and related strains from GenBank revealed that the newly determined sequences were related to the γ-Proteobacteria. Members of the genera Glaciecola and Alteromonas were the closest relatives of the six strains (87·890·8 % sequence similarity). The phylogenetic tree suggests that the isolates formed a separate cluster; this was supported by a bootstrap value of 100 % (Fig. 1).

(58K): Fig. 1. Phylogenetic tree based on almost-complete 16S rDNA sequences, comparing Agarivorans albus with members of Alteromonas and related genera. Numbers at branch-nodes are bootstrap values. Bar, 0·01 nucleotide substitution per position.

Morphological characteristics
Cells were 1·51·9 µm long and 0·70·9 µm wide with a single polar flagellum. They were Gram-negative rods that were straight or curved in shape (Fig. 2). All six isolates formed smooth, rounded colonies that were white in colour.

(117K): Fig. 2. Transmission electron micrograph of Agarivorans albus MKT 89 from growing liquid culture. Bar, 1 µm.

Biochemical and physiological characteristics
The six isolates exhibited almost-identical characteristics and all were able to hydrolyse agar. After incubating cultures for at least 1 week on marine agar at 23 °C, it was found that the agar began to liquefy; after several weeks, the agar was completely liquid. The strains did not grow anaerobically. All strains required NaCl for growth; no growth occurred in media that lacked NaCl or in the presence of 10 % NaCl.

In the BIOTEST system (Table 2), the six isolates exhibited oxidase, catalase and β-galactosidase activities and were able to reduce nitrate. On the other hand, lysine decarboxylase and ornithine decarboxylase activities were not detected. Agar and aesculin were hydrolysed, but hydrolysis of gelatin, arginine and urea was not observed. Fructose was utilized as a carbon source for strains MKT 82, 86, 87, 89 and 112, but not for MKT 106^T.

Table 2. Phenotypic characteristics that differentiate Agarivorans albus from previously described Alteromonas and Glaciecola species Taxa: 1, Agarivorans albus; 2, Alteromonas; 3, Glaciecola. +, Positive reaction or growth; , negative reaction or growth; V, variable reaction; NR, not reported.

Chemotaxonomic characteristics
The ubiquinone of the six strains was Q-8. The major fatty acids of the strains grown on marine broth are listed in Table 3. The predominant fatty acid of all six strains was non-polar. The major fatty acids of strains MKT 82, 86, 87, 106^T and 112 were C_{16 : 0}, C_{16 : 1}ω7c, C_{18 : 1}ω7c and C_{18 : 1}ω6c. Anteiso-C_{17 : 1}ω9c was the major fatty acid in MKT 89, but this was not common in the remaining five strains.

Table 3. Major fatty acids (%) of Agarivorans albus Taxa: 1, MKT 106T; 2, MKT 82; 3, MKT 86; 4, MKT 87; 5, MKT 89; 6; MKT 112. ND, Not detected.

DNA G+C content and DNADNA hybridization
The DNA G+C contents of the six strains ranged from 48·7 to 49·7 mol%. DNADNA relatedness values of each strain with the type strain (MKT 106^T) were 92·4 (MKT 82), 75·8 (MKT 86), 82·7 (MKT 87), 90·2 (MKT 89) and 80·7 (MKT 112) %. Levels of DNADNA relatedness among these six strains were all above 70 %. Phenotypic analysis gave results that enabled the isolates to be distinguished from the neighbouring genera Glaciecola and Alteromonas. The most distinctive characteristic of the isolates was their ability to hydrolyse agar; members of the genus Alteromonas hydrolyse gelatin, but not agar, and Glaciecola pallidula and Glaciecola punicea do not hydrolyse either agar or gelatin. An agar-hydrolysing species, Glaciecola mesophila, was reported recently (Romanenko et al., 2003). The novel isolates could also grow well in the absence of agar. They formed distinctive white colonies on marine agar 2216, whereas colonies of Glaciecola species ranged from pinkred or pale pink to having no pigment and those of Alteromonas species had no pigmentation. The isolates had a DNA G+C content of 4950 mol%, which was slightly higher than those of species of Glaciecola (4046 mol%) and Alteromonas (4447 mol%) (Bowman et al., 1998). Whole-cell fatty acid profiles of these six isolates were similar to those of species of Glaciecola and Alteromonas. However, C_{18 : 1}ω6c, which was one of the major fatty acids of the novel isolates (4·414·6 %), was not detected in species of Glaciecola or Alteromonas (Bowman et al., 1998). Phylogenetic analysis based on 16S rDNA sequences revealed that the six isolates formed a novel taxon. The genus Alteromonas, its closest relative, had 90·8 % 16S rDNA sequence similarity to the novel isolates.

The six isolates, which were isolated from four different species of marine creatures, formed a distinct group, exhibiting 99·4100·0 % 16S rDNA sequence similarity amongst themselves. Moreover, these strains shared 74·493·4 % DNADNA reassociation, which is clearly above the level of 70 % that is accepted as the limit for species relatedness (Wayne et al., 1987). These results demonstrate clearly that the six isolates belong to the same species.

One of the four species of marine creatures that were used as isolation sources was a tunicate; tunicates are known as seston-feeders. The other three species of molluscs are known as algae-feeders. One particular characteristic of the isolates was the ability to hydrolyse agar. Thus, the MKT strains are possibly important in the feeding habits of the marine creatures from which they were isolated.

In summary, on the basis of morphological, phenotypic and molecular data, the agar-degrading marine isolates can be classified as a novel genus and species, for which the name Agarivorans albus gen. nov., sp. nov. is proposed.

Description of Agarivorans gen. nov.
Agarivorans (A.ga.ri.vo'rans. N.L. neut. n. agarum agar; L. part. adj. vorans devouring, destroying; N.L. adj. agarivorans agar-devouring).

Gram-negative, strictly aerobic and agar-hydrolysing. Cells are rod-shaped and approximately 1·51·9x0·70·9 µm. Spores are not formed; motile by single polar flagella. No growth occurs without NaCl. Mesophilic. Hydrolysis of agar is observed. Major quinone is ubiquinone-8. Predominant cellular fatty acids are saturated and monounsaturated, straight-chain fatty acids. The genus is affiliated to the γ-Proteobacteria and presently contains one species, Agarivorans albus, which is the type species.

Description of Agarivorans albus sp. nov.
Agarivorans albus (al'bus. L. adj. albus white).

In addition to the characteristics that define the genus, the following characteristics are observed. Grows on marine agar 2216 and forms colonies with a smooth surface that are thin, circular in shape and white in colour. Major fatty acids are C_{16 : 1}ω7c, C_{16 : 0}, C_{18 : 1}ω7c and C_{18 : 1}ω6c. Other characteristics are listed in Table 2. Isolated from gastrointestinal tracts and attached internal organs of Mollusca.

The type strain, MKT 106^T (=IAM 14998^T=LMG 21761^T), was isolated from the marine mollusc Omphalius pfeifferi pfeifferi. DNA G+C content of the type strain is 49·5 mol%.

We are grateful to Aiko Hirata for providing skilful technical assistance in the production of transmission electron micrographs of the strains used in this study. We also thank Hans G. Trüper (University of Bonn, Bonn, Germany) for his help in the latinization of the new genus and species names.