FISHERIES SCIENCE 2002; 68: 703–705

Short Paper

Phylogenic analysis of three marine that have an ability to decompose Laminaria japonica

Motoharu UCHIDA,1,* Toshimichi MAEDA2 AND Tsuneo SHIBA2

1National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Kanagawa 236-8648 and 2Departmnent of Food Science and Technology, National Fisheries University, Shimonoseki, Yamaguchi 759-6595, Japan

KEY WORDS: 16S rDNA, Glaciecola, Laminaria-decomposing bacteria, phylogenic analysis, Pseudoalteromonas atlantica.

A large proportion of bacteria in coastal waters has ment, calculation of evolutionary distances by been reported to have a decomposing ability for Kimura’s two-parameter model,8 and construction macroalgae thallus and their polysaccharides.1,2 of a neighbor-joining phylogenic tree9 were per- On the other hand, bacterial isolates with a marked formed using the CLUSTAL W program.10 A boot activity to decompose algal polysaccharides are strap analysis of 1000 replicates was performed. expected to be enzyme sources of industrial use Sequences of 16S rDNA from the following bacte- for producing oligosaccharides with some useful ria were used in the phylogenic analysis: Pseudoal- bio-activities. teromonas atlantica (accession number of the Three bacterial strains, AR06, HA02, and TO01, DDBJ, EMBL, and GenBank nucleotide sequence were isolated from coastal waters in a previous databases; X82134), P.bacteriolytica (D89929), P. study1 and observed to have an ability to decom- carrageenovora (X82136), P.citrea (X82137), P.espe- pose Laminaria japonica and algal polysaccha- jiana (X82143), Alteromonas macleodii (X82145), A. rides.3 The strain AR06 shows the highest activity macleodii ssp. fijiensis (X85174), A. infernus among them under microscopic observation, (X85175), Shewanella putrefaciens (X82133), S. alga and can form single cell-algal detrital particles (Z95847), Colwellia psychroerythrus (U85842), C. from Laminaria4 and Ulva5 in an experimental rossensis (U14581), Marinomonas vaga (X67025), condition. In the present study, phylogenic analy- Glaciecola pallidula (U85854), G. punicea sis based on nucleotide sequence of 16S rRNA gene (U85853), Vibrio cholerae (X74695), Escherichia coli (16S rDNA) and DNA–DNA reassociation test were (J01859) and Oceanospirillum linum (M22365). conducted for strains AR06, HA02, and TO01. The 16S rDNA sequences of strain AR06, HA02, and Total DNA was extracted from the cultures of TO01 have been deposited in the DDBJ, EMBL the three strains using a commercial kit (G NOME, and GenBank database under accession numbers BIO101, CA, USA) and was used as a template for AB049728, AB049729, and AB049730, respectively. PCR amplification of 16S rDNA. The nearly full To prepare chromosomal DNA for the use in length 16S rDNA was amplified using primers 27F DNA–DNA reassociation test, bacterial cells were (5¢-dAGAGTTTGATCCTGGCTCAG-3¢) and 1492R suspended in Tris-EDTA buffer (pH 8.0) and treated (5¢-dTACGGTTACCTTGTTACGACTT-3¢).6 The com- with lysozyme and RNase (final concentration, 1 position of PCR mixture and the thermal profiles mg/mL and 0.1 mg/mL, respectively), and then were the same conditions as described in a previ- sodium dodecyl sulfate (final concentration, 2%) ous paper.7 The nucleotide sequence was deter- for lysis. The extracted DNA was purified by stan- mined using a Dye Terminator Cycle Sequencing dard method.11 The DNA–DNA reassociation Ready Reaction kit (Perkin Elmer, Boston, USA) test was conducted by a microplate hybridization and appropriate primers. Multiple sequence align- method12 with photobiotin labeling and colorimet- ric detection, using 1,2-phenylendiamine as the *Corresponding author: Tel: 81-45-788-7661. Fax: 81-45-788- substrate and streptavidine-peroxidase conjugate 5001. Email: [email protected] (Boehringer Mannheim Biochemica, IN, USA) as a Received 26 January 2001. Accepted 4 June 2001. colorimetric enzyme. 704 FISHERIES SCIENCE M Uchida et al.

The results of the phylogenic analysis showed the sequence similarity was found with Glaciecola that the three strains belonged to the g subclass of pallidula (95.2%). The genus Glaciecola was the class . Strain AR06 was included recently described by Bowman et al.13 and to date, in the genus Pseudoalteromonas (Fig. 1) and the includes only two (G. pallidula and G. highest percentage of the sequence similarity was punicea). Both species were isolated from Antarc- found with Pseudoalteromonas atlantica IAM tic ice-diatom assemblage and described to be psy- 12927T (99.9%). The DNA–DNA reassociation levels chrophilic.14 However, strain HA02 was isolated between the strains AR06 and the P.atlantica IAM from a boundary of subtropical and subarctic sea 12927T were 74 and 127% (Table1). Strain AR06 was regions,1 off Hakodate, and was not psychrophilic. assigned to P.atlantica based on these results and It is interesting that the two Glaciecola species and the phenotypic characteristics reported in a previ- strain HA02 are quite different in the isolation ous study.4 Strain HA02 was included in the genus source but common in that they have an ecologic Glaciecola (Fig. 1) and the highest percentage of relationship with algae. The sequence similarity of

Fig. 1 Phylogenic tree based on 16S rDNA sequence.

Table 1 DNA–DNA reassociation levels between the three Laminaria-decomposing bacteria and Alteromonas– Pseudoalteromonas species % Relatedness to labeled DNA from Strains AR06 HA02 TO01 IAM 12376T IAM 12640T IAM 12920T Strain AR06 100 31 11 127 34 12 Strain HA02 0 100 1 0 0 0 Strain TO01 0 35 100 27 8 16 Pseudoalteromonas atlantica IAM 12376T 74 2 0 100 18 0 Pseudoalteromonas espejiana IAM 12640T 36 41 0 49 100 1 Alteromonas macleodii IAM 12920T 0 34 19 5 12 100 Laminaria decomposing bacteria FISHERIES SCIENCE 705

the 16S rDNA 95.2% with the most closely related 5. Uchida M, Numaguchi K. Formation of protoplasmic G. pallidula strongly suggests that the strain HA02 detritus with characteristics favorable as food for secondary is a distinct species of Glaciecola although DNA– animals during microbial decomposition of Ulva pertusa DNA reassociation test is not conducted. Strain (Chlorophyta) frond. J. Mar. Biotechnol. 1996; 4: 200– 206. TO01 was included in the genus Alteromonas (Fig. 6. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S riboso- 1) and the highest percentage of the sequence mal DNA amplification for phylogenic study. J. Bacteriol. similarity was found with Alteromonas macleodii 1991; 173: 697–703. (98.9%). The DNA–DNA reassociation values 7. Maeda T, Yoshinaga I, Murakami M, Shiba T, Ishida Y. between the strains TO01 and A. macleodii IAM Growth and phylogenic characteristics of a pelagic marine 12920T were 16–19% (Table1) and it was suggested bacterium, Alteromonas sp. KE10, adapted to low-nutrient that strain TO01 is a distinct species in the genus environments. Microbes Environ. 1999; 14: 209–217. Alteromonas. 8. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of We are grateful to Drs Aritsune Uchida and Masa- nucleotide sequences. J. Mol. Biol. 1980; 16: 111–120. taka Satomi for their technical suggestions in con- 9. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenic trees. Mol. Biol. Evol. ducting the phylogenic analysis and DNA–DNA 1987; 4: 406–425. reassociation test. This study was supported in part 10. Thompson JD, Higgins DG, Gibson TJ, Clustal W. Improving by a grant (the PIONEER Research Project Fund the sensitivity of progressive multiple sequence alignment Grant No. PRPF1204) from the Research Council of through sequence weighting, position-specific gap penal- the Ministry of Agriculture, Forestry, and Fisheries, ties and weight matrix choice. Nucl. Acids Res. 1994; 22: Japan. 4673–4680. 11. Saito H, Miura K. Preparation of transforming deoxyri- bonucleic acid by phenol treatment. Biochem. Biophys. Acta 1963; 72: 619–629. REFERENCES 12. Ezaki T, Hashimoto Y, Yabuuchi E. Fluorometric deoxyri- bonucleic acid-deoxyribonucleic acid hybridization in 1. Uchida M, Nakayama A. Isolation of Laminaria-frond microdilution wells as an alternative to membrane filter decomposing bacteria from Japanese coastal waters. hybridization in which radioisotopes are used to determine Nippon Suisan Gakkaishi 1993; 59: 1865–1871. genetic relatedness among bacterial strains. Int. J. Syst. Bac- 2. Uchida M, Nakayama A, Shinnichiro A. Distribution and teriol. 1989; 39: 224–229. characterization of bacteria capable of decomposing brown 13. Bowman JP, McCammon SA, Brown JL, McMeekin TA. algae fronds in waters associated with Laminaria vegeta- gen. nov., sp. nov. and Glaciecola pal- tion. Fish. Sci. 1995; 61: 117–120. lidula gen. nov., sp. nov.: Psychrophilic bacteria from 3. Uchida M. Enzyme activities of marine bacteria involved in Antarctic sea-ice habitats. Int. J. Syst. Bacteriol. 1998; 48: Laminaria-thallus decomposition and the resulting sugar 1213–1222. release. Mar. Biol. 1995; 123: 639–644. 14. Bowman JP, McCammon SA, Brown MV, Nichols DS, 4. Uchida M. Formation of single cell detritus densely covered McMeekin TA. Diversity and association of psychrophilic with bacteria during experimental degradation of Lami- bacteria in Antarctic sea ice. Appl. Environ. Microbiol. 1997; naria japonica thalli. Fish. Sci. 1996; 62: 731–736. 63: 3068–3078.