International Journal of Systematic and Evolutionary Microbiology (2000), 50, 901–907 Printed in Great Britain Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov. Elena P. Ivanova,1,2 Ludmila A. Romanenko,2 Jongsik Chun,1† Maria H. Matte,1,3 Glavur R. Matte,1,3 Valery V. Mikhailov,2 Vasilii I. Svetashev,2 Anwarul Huq,1,5 Tim Maugel4 and Rita R. Colwell1,5 Author for correspondence: Rita R. Colwell. Tel: j1 703 306 1000. Fax: j1703 306 0109. e-mail: colwell!umbi.umd.edu 1 Center of Marine Two bacterial strains, KMM 227T and 231T, were isolated from seawater Biotechnology, University samples collected from the north-western Pacific Ocean at a depth of of Maryland Biotechnology Institute, 4000–5000 m and were characterized using polyphasic taxonomy. Both were 701 E. Pratt St, Baltimore, Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for MD 21202, USA growth (06–150%). The temperature for growth was 4–30 SC. Both strains 2 Pacific Institute of were rod-shaped, with a single flagellum. However, strain KMM 231T revealed Bioorganic Chemistry of a single long fimbrium. Cellular fatty acids detected in the isolates were the Far-Eastern Branch of the Russian Academy of predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. Sciences, 690022 70%). Also present were saturated and monounsaturated straight-chain fatty Vladivostok, Pr. 100 Let acids. Results of phylogenetic analyses, employing three tree-making methods, Vladivostoku 159, Russia strongly indicated that the two strains formed a distinct lineage within a clade 3 School of Public Health, containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the University of Sao Paulo, Av. D. Arnaldo, 715, γ-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 012466904, Brazil 969% and genomic DNA relatedness of 27%; the latter was determined by 4,5 Department of Zoology4, dot-blot hybridization. The strains were differentiated by the presence of and Department of Cell fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG Biology and Molecular profiles. Given the polyphasic evidence accumulated in this study, it is Biology (CBMB)5, University of Maryland, proposed that the two deep-sea isolates be classified in the genus Idiomarina College Park, MD 20742, gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and USA Idiomarina zobellii sp. nov. (type strain is KMM 231T). Keywords: deep-sea bacteria, new genus, new species, Idiomarina abyssalis, Idiomarina zobellii INTRODUCTION isolated from seawater, algae and marine inverte- brates (Baumann & Baumann, 1981; Baumann et al., Marine bacteria belonging to Alteromonas, Pseudo- 1984; Gauthier & Breittmayer, 1992; Gauthier et al., alteromonas, Marinomonas, Halomonas and Oceano- 1995; Dobson & Franzmann, 1996). Identification of spirillum have been characterized as aerobic, Gram- these oxidative marine bacteria at the species level negative, heterotrophic, mainly rod-shaped organisms remains difficult and time consuming, although members of the five genera cited above have pheno- ................................................................................................................................................. typic and chemotaxonomic features that differentiate † Present address: Korean Collection for Type Cultures, Korea Research them at the genus level. Representatives of these micro- Institute of Bioscience and Biotechnology, PO Box 115, Yusong, Taejon 305- 600, Republic of Korea. organisms are interesting in terms of their metabolic The GenBank accession numbers for the 16S rDNA sequences of Idiomarina characteristics, psychrophilic nature, halotolerance abyssalis KMM 227T and Idiomarina zobellii KMM 231T are AF052740 and and ubiquitous distribution in the marine environ- AF052741, respectively. ment. 01182 # 2000 IUMS 901 E. P. Ivanova and others (a) (b) (c) (d) (e) ................................................................................. Fig. 1. Negatively stained cells of Idiomarina abyssalis KMM 227T(a, b, c) and Idiomarina zobellii KMM 231T (d, e). (a) Cell with single flagellum on one pole and outer sheath-like structure ballooning over one end; (b) KMM 227T in ex- ponential phase of growth; (c) outer sheath-like structure; (d, e) cells of KMM 231T with fimbria, along with a flagellum at one pole. (a, b) Bar, 1 µm; (c, d, e) bar, 0n5 µm. The presence of as yet undescribed novel bacterial taxa macleodii ATCC 27126T, Vibrio cholerae ATCC 14035T, T in a variety of extreme marine environments is in- Aeromonas jandaei ATCC 49568 , Marinomonas communis ATCC 27118T, Pseudoalteromonas haloplanktis subsp. halo- dicative of the fact that they represent a resource of T microbial diversity (Deming et al., 1988; Gauthier et planktis IAM 12915 and Escherichia coli ATCC 25922 were al., 1992; Irgens et al., 1996; Bowman et al., 1997a, b; employed as reference strains. Bozal et al., 1997; Ra' gue! nes et al., 1997). Charac- Phenotypic characterization. Standard methods developed terization of two deep-sea isolates is reported here and, for characterization of Alteromonas-like species were per- formed, as described by Baumann et al. (1972), Baumann & based on the polyphasic evidence obtained, it is Baumann (1981), Smibert & Krieg (1994), and Ivanova et al. proposed that they be classified as Idiomarina abyssalis (1996, 1998). The following physiological and biochemical sp. nov. and Idiomarina zobellii sp. nov., in a new properties were examined: oxidation\fermentation of glu- genus, Idiomarina gen. nov. cose; arginine dihydrolase; accumulation of poly-β-hydroxy- butyrate; cell pigmentation; cell morphology; Gram stain- ing; motility; sodium requirement; oxidase and catalase METHODS production; and the ability to hydrolyse gelatin, agar, DNA, Sampling and isolation. Water samples were collected from a starch, Tween-80 and chitin. Anaerobic growth was tested using the Oxoid Anaerobic system. The requirement for Na+ depth of 4000–5000 m (salinity, 34=; temperature, 2 mC) in ions was studied on medium that contained (w\v): 0 25% the north-western area of the Pacific Ocean (latitude 8m20 N, n yeast extract, 0 1% glucose, 0 02% K HPO ,0005% longitude 133m0 W) during July, 1985. The deep water n n # % n samples were collected using a standard hydrological plastic MgSO%.7H#O (pH 7n8). Salt tolerance tests were performed bathymeter. Isolation of the strains was achieved at atmos- on Trypticase Soy Agar (TSA; Difco) with NaCl concen- trations of 0 6–20 0% (w\v). The temperature range for pheric pressure by plating 0n1 ml seawater onto oligotrophic n n agar prepared with full-strength seawater, amended with growth was determined on TSA containing 3% (w\v) NaCl; plates were incubated for 10 d at 4, 10, 15, 20, 25, 30 0n3% (w\v) Bacto Peptone (Difco) and 1n5% (w\v) Bacto Agar (Difco). The strains were subsequently purified on and 37 mC. The pH range for growth was determined in medium B, containing 0 2% (w\v) Bacto Peptone, 0 2% Marine Broth with the pH values of separate batches of n n media adjusted to 4, 4 5, 5, 6, 7, 8, 8 5, 9, 9 5 and 10. The pH (w\v) casein hydrolysate (Merck), 0 2% (w\v) Bacto Yeast n n n n was adjusted with 10 M NaOH or HCl. Extract (Difco), 0n1% (w\v) glucose, 0n002% (w\v) KH#PO%,0n005% (w\v) MgSO% and 50% (v\v) natural Electron micrographs of negatively stained cells, using seawater, as described elsewhere (Ivanova et al., 1996). 1n25% uranyl acetate, were obtained using a Zeiss EM 10 The strains were maintained on a semisolid B medium, CA electron microscope (80 kV). under mineral oil, at 4 mC and at k80 mC in Marine Broth Utilization of various organic substrates, including amino (Difco) supplemented with 30% (v\v) glycerol. Alteromonas acids, at 0n1% (w\v) as sole carbon source was performed 902 International Journal of Systematic and Evolutionary Microbiology 50 Idiomarina gen. nov. Table 1. Characteristics differentiating Idiomarina species from related genera ................................................................................................................................................................................................................................................................................................................. Data obtained from this and earlier studies (D’Aoust & Kushner, 1972; Baumann at al., 1984; Krieg, 1984; Vreeland, 1984; Deming et al., 1988; Bertone et al., 1996; Bowman et al., 1998). j, Trait present; k, trait absent; , varies between strains; , not determined. Strains or genera: 1, I. abyssalis KMM 227T;2,I. zobellii KMM 231T;3,Alteromonas;4,Colwellia; 5, Halomonas;6,Marinomonas;7,Oceanospirillum;8,Pseudoalteromonas. Characteristic Strain/genus 1 2 3 4 5 678 Cell diameter (µm) 0n7–0n90n7–0n90n7–1n00n5–1n00n6–1n10n7–1n50n3–1n40n2–1n5 Flagella arrangement: Polar j j j j j kjj Bipolar k k k k k jjk Lateral k k k k j kkj Organelles k Fimbria kkFilaments kkk Metabolism Aerobic Aerobic Aerobic Facultative Aerobic Aerobic Aerobic Aerobic anaerobic Growth occurs: in NaCl (%) 0n6–15 1–10 1–6 "2n5 0–20 1–6 0n5–10 1–9 at 4 mC jjkj kk Presence of: Poly-β-hydroxybutyrate kkk j kjk Gelatinase, lipase j j j j k kkj Chitinase kj jkkk Acid produced from sugars k k j j j jkj Utilization of: -Glucose k k j j j jkj -Fructose kkj jjk Mannitol kkjjk Sucrose kkj jkk Maltose kkjjjv k Lactose kkj j kkk Major fatty acid type Iso-branched Iso-branched