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1 Institute for Biological Resources and Function, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan
2 Laboratory of Electron Microscopy, School of Dentistry, Hokkaido University, Kita-ku, Sapporo 060-8586, Japan
Correspondence
Isao Yumoto
i.yumoto{at}aist.go.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MD17T is AB094794.
A table showing DNA–DNA relatedness among strains examined in this study and micrographs showing the morphology of strain MD17T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Arpigny et al., 1993; Choo et al., 1998; Rashid et al., 2001). However, few cold-adapted bacteria that degrade lipids at low temperatures have been identified at the species level from taxonomic viewpoints.
We have been isolating and characterizing novel cold-adapted micro-organisms from a cold current in the Okhotsk Sea (Yumoto et al., 1998; Kawasaki et al., 2002). It is expected that there are novel cold-adapted micro-organisms that can degrade lipids at low temperatures. In our screening for isolates that can grow at 0 °C on marine agar 2216 and that exhibit lipase activity at 5 °C, we isolated a facultative psychrophile, strain MD17T, which was classified phylogenetically in the genus Psychrobacter (based on 16S rRNA gene analysis), from a sea-water sample obtained from the coast of the Okhotsk Sea in Hokkaido, Japan, when a cold current carrying drifting ice from the east coast of Sakhalin, Russia, flowed into the area. In this study, we examined the phenotypic and chemotaxonomic characteristics and the phylogenetic position of the isolate and found that the strain should be classified as a novel species that belongs to the genus Psychrobacter.
In February 1998, sea water was collected from the Monbetsu coast (44° 31' N 143° 39' E), Hokkaido, Japan, which opens to the Okhotsk Sea. The temperature of the sea-water sample was -1 °C. The sea-water sample was spread directly on plates of marine agar 2216 (Difco) and incubated at 0 °C for 4 weeks. Colonies that appeared were transferred to blue agar (Difco) and incubated at 15 °C in order to confirm lipase production. Colonies that exhibited lipid digestion were further incubated at 5 °C in an olive oil-containing liquid medium (1 % olive oil, 50 mM Tris, 190 mM NH4Cl, 0·33 mM K2HPO4 and 0·1 mM FeSO4.7H2O in 1000 ml of 50 % Herbst's artificial sea water). Herbst's artificial sea water contains the following (l distilled water)-1: NaCl, 30 g; KCl, 0·7 g; MgSO4.7H2O, 5·3 g; CaSO4.2H2O, 1·3 g; and MgCl2.6H2O, 10·8 g. Strain MD17T was selected as a micro-organism that could grow at 0 °C and degrade lipids at 5 °C.
Cells for chemotaxonomic analysis were harvested 2 days after cultivation with shaking (140 r.p.m.) at 15 °C. In addition to the isolate, Psychrobacter immobilis ATCC 43116T, Psychrobacter glacincola ATCC 700754T, Psychrobacter faecalis DSM 14664T, Psychrobacter proteolyticus DSM 13887T, Psychrobacter marincola DSM 14160T and Psychrobacter submarinus DSM 14161T were used as reference strains for DNA–DNA hybridization. Micro-organisms were cultivated in marine broth 2216 (Difco) with shaking (140 r.p.m.) at 15 °C for 2 days.
Marine broth or agar 2216 (Difco) was used as the basal medium for aerobic cultivation unless otherwise stated. The culture was incubated at 15 °C for 3 weeks and experiments were performed at least twice. Morphological, physiological and biochemical tests were performed as described by Barrow & Feltham (1993). Carbohydrate metabolism was tested according to the method of Leifson (1963). The result was checked daily for 3 weeks after inoculation. For hydrolysis of macromolecular substances, PYB agar (pH 7·5) that contained (per litre of 50 % Herbst's artificial sea water): 5 g polypeptone (Nihon Pharmaceutical), 1 g yeast extract (Kyokuto), 15 g agar and 0·5–1·0 % substrate was used as the basal medium. Alginase activity was determined by flooding a 3-week culture of the isolate on PYB agar plates that contained 0·5 % alginic acid with 70 % ethanol. Various substrates were tested as sole sources of carbon and energy in USTM medium (pH 7·5), which contained 0·2 % substrate, 50 mM Tris, 190 mM NH4Cl, 0·33 mM K2HPO4 and 0·1 mM FeSO4.7H2O per litre of 50 % Herbst's artificial sea water. Media that lacked a carbon source were prepared as negative controls to account for any background growth.
The optimum growth temperature of strain MD17T was determined by using a temperature gradient incubator (model TN-2612; Advantec). The temperature gradient range used was 5–35 °C. L-Shaped tubes that contained 10 ml marine broth 2216 were used.
Analyses of whole-cell fatty acids and isoprenoid quinones were performed as described previously (Yumoto et al., 2001).
DNA was prepared from bacterial cells according to the method of Marmur (1961). The G+C content of the DNA was determined according to the method of Tamaoka & Komagata (1984). Levels of DNA relatedness were determined fluorometrically by the method of Ezaki et al. (1989) by using photobiotin-labelled DNA probes and microplates.
The sequence that corresponded to positions 27–1519 in the 16S rRNA gene of Escherichia coli (Brosius et al., 1978) was amplified by PCR. The 1·5 kb PCR product was sequenced directly by the dideoxynucleotide chain-termination method, using an ABI PRISM 377 DNA sequencer (Applied Biosystems). Multiple alignments of the sequences were performed, nucleotide substitution rates (Knuc values) were calculated and a neighbour-joining phylogenetic tree (Kimura, 1980; Saitou & Nei, 1987) was constructed by using the CLUSTAL W program (Thompson et al., 1994) with the determined 1492 bp sequence. Similarity values between the sequences were calculated by using the GENETYX program (Software Development).
Morphological, physiological and biochemical characteristics of strain MD17T are given in the species description. The strain was able to hydrolyse only Tweens and lipids among the macromolecular compounds tested. It did not utilize carbohydrates. Its generation times at 16 and 5 °C were 216 and 504 min, respectively. These values are longer than those of the obligate psychrophile Moritella marina: 80·7 min at 15 °C and 226 min at 3 °C (Morita & Albright, 1965).
GLC analysis of the fatty acids of strain MD17T revealed that the major components were C10 : 0 (2·9 %), C12 : 0 (2·4 %), C16 : 19c (9·4 %), C17 : 1 (21·5 %), C18 : 19c (57·4 %) and C18 : 111c (2·3 %). Isoprenoid quinones isolated from strain MD17T by using TLC were analysed by HPLC. Analysis revealed that ubiquinone-8 (Q-8) was the predominant isoprenoid quinone in the strain.
The almost-complete 16S rRNA gene sequence of strain MD17T, which consisted of 1492 nt, was found to have 99·0 and 98·7 % similarity to those of P. glacincola DSM 12194T and P. immobilis DSM 7229T, respectively. The sequence of strain MD17T also exhibited high similarity (97·0, 97·4, 97·5 and 98·0 %) to P. faecalis DSM 14664T, P. marincola DSM 14160T, P. submarinus DSM 14161T and P. proteolyticus DSM 13887T, respectively. Phylogenetic analysis based on 16S rRNA gene sequences also showed that the isolate is a member of the genus Psychrobacter and is related closely to P. glacincola DSM 12194T and P. immobilis DSM 7229T (Fig. 1).
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Furthermore, several growth and phenotypic characteristics of strain MD17T were distinct from those of other related Psychrobacter species (Table 1).
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; Bowman et al., 1996, 1997a, b; Maruyama et al., 2000). Our results also indicate that members of the genus Psychrobacter are successful in colonizing low-temperature environments. It is ecologically interesting that phylogenetically and phenotypically related groups of bacteria are distributed over a wide variety of cold environments. The ability to grow rapidly at low temperatures, halotolerance and variation of substrate specificity of Psychrobacter species may be among the reasons for their wide distribution in cold environments. On the basis of phenotypic and chemotaxonomic characteristics, phylogenetic position based on 16S rRNA gene analysis and relatedness by DNA–DNA hybridization, strain MD17T was confirmed to be a member of a novel species that belongs to the genus Psychrobacter, for which the name Psychrobacter okhotskensis sp. nov. is proposed.
Description of Psychrobacter okhotskensis sp. nov.
Psychrobacter okhotskensis (ok.hot.sken'sis. N.L. adj. okhotskensis from Okhotsk Sea, the place where the micro-organism was isolated).
Cells are coccobacilli (0·7–1·0x0·8–1·3 µm), Gram-negative and without a flagellum. Colonies are circular, convex and colourless, with an entire margin. The species is positive for catalase and oxidase. It does not produce acid from L-arabinose, D-glucose, D-fructose, D-maltose, D-mannose, melibiose, sucrose, D-xylose, raffinose, myo-inositol, mannitol, sorbitol, D-galactose, trehalose, lactose or D-cellobiose under aerobic conditions. Growth occurs in medium supplemented with 0–10 % NaCl, but not in medium with salinity higher than 12 %. Growth occurs between 0 and 35 °C, but not at temperatures higher than 36 °C; optimum growth temperature is 25 °C. The organism is positive for the reduction of nitrate to nitrite and indole production, but negative for urease, Voges–Proskauer, methyl red and ONPG tests. Lipid and Tweens 20, 40, 60 and 80 are hydrolysed, but casein, gelatin, DNA, alginic acid, hippurate and aesculin are not. The organism utilizes 3-hydroxybutyrate, pyruvate, L-malate and pimelate as sole carbon and energy sources for growth, but not L-arabinose, D-glucose, D-fructose, glycerol, lactose, D-maltose, D-mannose, melibiose, raffinose, sucrose, D-xylose or sorbitol. Whole-cell fatty acids consist predominantly of C10 : 0, C12 : 0, C16 : 19c, C17 : 1, C18 : 19c and C18 : 111c. Major isoprenoid quinone is Q-8. G+C content of DNA is 46·7 mol%, as determined by HPLC.
The type strain is strain MD17T (=NCIMB 13931T=JCM 11840T). Strain MD17T was isolated from sea water from the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan, when ice carried by a cold current came to the area.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Barrow, G. I. & Feltham, R. K. A. (editors) (1993). Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edn. Cambridge: Cambridge University Press.
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