International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1291–1297 DOI: 10.1099/ijs.0.02087-0 Psychrobacter submarinus sp. nov. and NOTE Psychrobacter marincola sp. nov., psychrophilic halophiles from marine environments 1 Pacific Institute of Lyudmila A. Romanenko,1 Peter Schumann,2 Manfred Rohde,3 Bioorganic Chemistry, 4 1 2 Far-Eastern Branch, Anatoly M. Lysenko, Valery V. Mikhailov and Erko Stackebrandt Russian Academy of Sciences, 690022 Vladivostok, Prospekt 100 Author for correspondence: Erko Stackebrandt. Tel: j49 531 2616 352. Fax: j49 531 2616 418. Let Vladivostoku, 159, e-mail: erko!dsmz.de Russia 2,3 DSMZ – Deutsche Sammlung von Two novel psychrophilic, halophilic, Psychrobacter-like bacteria, strains KMM Mikroorganismen und 225T and KMM 277T, were isolated from sea water and the internal tissues of an 2 Zellkulturen GmbH and ascidian Polysyncraton sp. specimen, respectively, and characterized using a GBF – Gesellschaft fu$ r Biotechnologische polyphasic approach, which included phenotypic, genotypic, chemotaxonomic Forschung GmbH3 , and phylogenetic analyses. The novel marine isolates were Gram-negative, D-38124 Braunschweig, aerobic, coccoid, oxidase- and catalase-positive, non-pigmented, non-motile, Germany psychrophilic and halophilic and they utilized a restricted spectrum of carbon 4 Institute of Microbiology, sources. Strains KMM 225T and KMM 277T required sea water or sodium ions for Russian Academy of Sciences, 117811 Moscow, growth and were tolerant of up to 12–15% (w/v) NaCl. Growth of strains KMM Russia 225T and KMM 277T was observed at 4–35 and 7–35 SC, respectively. The DNA GMC contents of KMM 225T and KMM 277T were respectively 468 and 507 mol%. Comparison of almost complete 16S rDNA sequences of strains KMM 225T and KMM 277T revealed that both strains were phylogenetically most closely related to each other (999% sequence similarity) and slightly less related to Psychrobacter glacincola, with 972 and 978% similarity, respectively. DNA–DNA reassociation between KMM 225T and KMM 277T revealed 15% similarity, whereas similarity to other Psychrobacter species was 14–25%. Strains KMM 225T and KMM 277T differed from one another in their growth temperature, organic substrate utilization, antibiotic sensitivity and DNA GMC content. Both strains examined could be distinguished from all previously described Psychrobacter species by their physiological, genotypic and phylogenetic characteristics. On the basis of the physiological and molecular properties of the novel isolates, the names Psychrobacter submarinus sp. nov. (type strain KMM 225T l DSM 14161T) and Psychrobacter marincola sp. nov. (type strain KMM 277T l DSM 14160T) are proposed. Keywords: Psychrobacter submarinus sp. nov., Psychrobacter marincola sp. nov., psychrophilic, halophilic bacteria, marine environments Gram-negative, aerobic, non-motile, psychrophilic, tive, non-motile, psychrophilic or psychrotolerant, halotolerant micro-organisms are common inhabi- halotolerant bacteria (Juni & Heym, 1986). DNA– tants of terrestrial and marine environments, including rRNA hybridization analysis performed by Rossau et the Antarctic and deep oceanic environments. The al. (1991) showed that this genus belongs to the family genus Psychrobacter was described to accommodate Moraxellaceae, which also includes the genera Gram-negative, moraxella-like, coccoid, oxidase-posi- Moraxella and Acinetobacter (Rossau et al., 1991). Members of the genus Psychrobacter have been iso- ................................................................................................................................................. lated from a wide range of habitats including sea The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences water, the gills and skin of fish, poultry, food and of strains KMM 225T and KMM 227T are respectively AJ309940 and clinical sources (Juni, 1991). Recently, three Antarctic AJ309941. Psychrobacter species with low growth-temperature 02087 # 2002 IUMS Printed in Great Britain 1291 L. A. Romanenko and others optima were described, Psychrobacter urativorans and Psychrobacter frigidicola, both from Antarctic ornitho- genic soils, and Psychrobacter glacincola from Ant- arctic sea ice (Bowman et al., 1996, 1997), indicating that members of the genus Psychrobacter are wide- spread in cold Antarctic environments. The novel species Psychrobacter pacificensis, which differs from known terrestrial and Antarctic species, was described for psychrophilic strains isolated from deep sea water (5000–6000 m) of the Japan Trench (Maruyama et al., 2000). In the present study, the taxonomic position of two strains, KMM 225T and KMM 227T, from marine environments was defined. Results revealed that these bacteria represent two novel species of the genus Psychrobacter, for which the names Psychrobacter submarinus sp. nov. and Psychrobacter marincola sp. nov. are proposed. Isolation, cultural characteristics and morphology Strain KMM 225T was isolated from a sea-water sample obtained at a depth of 300 m in the north- western part of the Pacific Ocean (12m 41h N, 132m 38h W) in July 1985. The sea-water sample was taken with a plastic hydrological bathometer. Strain KMM 277T was isolated from the homogenized internal tissues of the ascidian Polysyncraton sp., collected in the Indian Ocean (33m 41h S, 134m 28h E) in March 1988. The homogenate was prepared and diluted in sterile sea water. Aliquots of the sea-water sample and the diluted homogenate were spread on agar plates of sea-water medium (SWM), which was made up of 750 ml sea water, 250 ml distilled water and the following com- −" ponents (g l ): peptone (5n0), yeast extract (2n5), glucose (1n0), K#HPO% (0n2), MgSO% (0n05) and agar (15n0). Samples were incubated for 7–10 days at 28 mC. Isolates were cultivated aerobically on SWM, 2216 marine agar (MA) or marine broth (MB) (Difco) at 25–28 mC. Bacteria were stored at k80 mCin30% (v\v) glycerol. Isolates designated KMM 225T and T KMM 277 have been deposited in the Collection of ................................................................................................................................................. Marine Micro-organisms (KMM) of the Pacific In- Fig. 1. Transmission electron micrographs showing general stitute of Bioorganic Chemistry, Vladivostok, Russia. morphology of negatively stained cells of Psychrobacter sub- marinus KMM 225T (a, b) and Psychrobacter marincola KMM For determination of biochemical and cultural tests, 277T (c). Bars, 1 (a), 0 5 (b) and 0 25 (c) µm. except those used for determination of the temperature n n range for growth, Psychrobacter immobilis CIP 102557T, P. urativorans CIP 105100T and P. frigidicola CIP 105101T were cultivated on trypticase soy agar (TSA; Difco) at 28, 20 and 15 mC, respectively. uranyl acetate and carbon film. Samples were Psychrobacter phenylpyruvicus CIP 82.27T was grown examined with a Zeiss transmission electron micro- on brain\heart infusion agar (BHIA; Difco) supple- scope TEM910 at an acceleration voltage of 80 kV at mented with 5% (v\v) blood serum at 30 mC and P. calibrated magnifications. Motility was observed with glacincola CIP 105313T was grown on MA 2216 at a light microscope by the hanging-drop method. 15 mC. The strains studied were slightly different in mor- Cell morphology was examined by transmission elec- phology and cell dimensions. Whereas cells of KMM T tron microscopy of exponential-phase cells grown in 225 were ovoid, 1n6–1n9 µm long and 0n7–1n0 µmin MB 2216. Cells were fixed with 1% (v\v) glutaral- diameter (Fig. 1a, b), those of KMM 277T were dehyde and negatively stained with 4% (w\v) aqueous coccoid, 0n7–1n0 µm in diameter (Fig. 1c). 1292 International Journal of Systematic and Evolutionary Microbiology 52 Two novel Psychrobacter species Growth characteristics and metabolic properties reduction, tryptophan deaminase, phenylalanine deaminase, the ONPG test, Simmons’ citrate and The Gram-reaction, oxidase, catalase, production of Voges–Proskauer tests, H S production, denitri- caseinase, deoxyribonuclease, gelatinase and lipase # fication, acid production from -glucose, -xylose, (Tween 80) were tested according to the standard rhamnose, galactose, maltose, lactose and mannitol methods described by Smibert & Krieg (1994) using and utilization of citrate, -malate, lactate, acetate, SWM as the basal medium. Amylase activity was propionate, -histidine, -proline, -alanine and n- observed on nutrient medium supplemented with valerate. starch at a concentration of 10% (w\v) by flooding plates with 1% (w\v) iodine solution. The ability to grow at different temperatures was tested on MA and Chemotaxonomy TSA at 4, 7, 10, 15, 25, 30, 35, 37 and 40 mC. The pH Cellular fatty acid methyl esters obtained from cells range for growth (5n0–10n0) was tested using MB with grown in MB at 28 mC by the method of Stead et al. pH values adjusted by the addition of 5 M NaOH or (1992) were separated by GC (Groth et al., 1996) and HCl. Sodium ion requirement and tolerance of NaCl identified as described previously (Schumann et al., were examined on SWM prepared with the artificial 1997). Polar lipids extracted by the method of sea-water base adjusted so that the amount of NaCl Minnikin et al. (1979) were identified by two- was between 0 and 20% (w\v). Oxidative utilization of dimensional TLC and spraying with specific reagents glucose was determined by the method of Leifson (Collins & Jones, 1980). The novel isolates possessed (1963). Leifson’s medium was also used for testing acid