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Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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-Proteobacteria, designated strain BH030046T, was isolated from a solar saltern in Korea. Cells were Gram-negative, chemoheterotrophic, short rod-shaped and motile with a polar flagellum. Comparative 16S rRNA gene sequence analysis revealed that strain BH030046T formed a distinct lineage in the family Alteromonadaceae and was distinguished from its closest related genera Alteromonas (91·4–94·8 %), Aestuariibacter (92·1–93·5 %) and Glaciecola (92·1–93·5 %) on the basis of low 16S rRNA gene sequence similarities. Physiological and biochemical data also showed that the isolate was different from members of these three genera. The predominant cellular fatty acids were C16 : 0 and C18 : 1
7c. DNA G+C content was 48 mol% and the major isoprenoid quinone was Q-8. The strain grew optimally at 30–35 °C, pH 7·0–8·0 and 2–5 % NaCl. On the basis of physiological and molecular properties, strain BH030046T (=KCTC 12239T=DSM 16280T) represents a novel genus and species in the family Alteromonadaceae, for which the name Salinimonas chungwhensis gen. nov., sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BH030046T is AY553295.
A transmission electron micrograph of the general morphology of a cell of strain BH030046T, a maximum-likelihood tree of the phylogenetic relationships of the strain and related taxa and a table detailing its fatty acid composition are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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combined the genera Alteromonas, Pseudoalteromonas, Idiomarina and Colwellia into a new family Alteromonadaceae belonging to the -Proteobacteria, new members of the family Alteromonadaceae such as the genera Glaciecola, Thalassomonas and Aesturiibacter have been isolated from samples from a wide range of marine habitats (Bowman et al., 1998; Macián et al., 2001; Yi et al., 2004). They are usually Gram-negative, heterotrophic, motile bacteria with a polar flagellum. In the screening of halophilic micro-organisms, a non-spore-forming rod bacterium (strain BH030046T) with a polar flagellum belonging to the family Alteromonadaceae was isolated. The aim of this study was to determine the taxonomic status of this strain using a polyphasic approach. Strain BH030046T formed a novel genus and species in the family Alteromonadaceae, for which the name Salinimonas chungwhensis gen. nov., sp. nov. is proposed. Strain BH030046T was isolated from a solar saltern in the Chungwha area on the Yellow Sea in Korea. Soil samples were serially diluted with 1 % (w/v) saline solution and spread on marine agar 2216 (MA) (Difco) with the addition of 5 % (w/v) NaCl [final concentration 6·94 % NaCl (w/v)] and incubated for 2 days at 35 °C. Requirement for and tolerance of NaCl were determined in nutrient broth (NB; 3·0 g beef extract and 5·0 g peptone l–1) (Difco) supplemented with modified artificial sea water [ASW; 0–30 % (w/v) NaCl, 5·94 g MgSO4.7H2O, 4·53 g MgCl2.6H2O, 0·64 g KCl and 1·3 g CaCl2 l–1). The isolate was routinely grown aerobically on MA for 2 days at 35 °C except where indicated otherwise. Growth was tested at different temperatures (4–55 °C) on MA and at different pH values (5·0–10·0) in NB containing ASW with 3 % (w/v) NaCl.
Cell morphology was studied using light microscopy and transmission electron microscopy (TEM). Motility was observed after 12 and 36 h in wet mounts using a light microscope (Nikon E600). The flagellum type was determined by TEM using cells from the exponential growth phase. Cells were mounted on Formvar-coated copper grids and negatively stained with 1 % (w/v) potassium phosphotungstate (pH 7·0). Grids were examined in a transmission electron microscope (JEOL JEM-1010) operated at 60 kV.
For quantitative analysis of whole-cell fatty acids, strain BH030046T was cultivated on MA for 2 days at 35 °C. Fatty acid methyl esters were prepared according to the instructions of the Microbial Identification System (MIDI; Microbial ID, Inc.) and analysed using GC/MS.
Catalase activity was determined by bubble production in 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Nitrate reduction and hydrolysis of aesculin, casein, starch, Tween 80, urea, hypoxanthine, tyrosine, gelatin and xanthine were determined on MA according to the methods of Cowan & Steel (1965), Lanyi (1987) and Smibert & Krieg (1994). Acid production from carbohydrates was tested as described by Leifson (1963); all suspension media were supplemented with ASW containing 3 % (w/v) NaCl. Enzyme activities were determined using the API ZYM system (bioMérieux) according to the manufacturer's instructions, with the suspended cells in ASW with 3 % (w/v) NaCl.
Isoprenoid quinones were analysed as described by Komagata & Suzuki (1987) using HPLC fitted with a reversed-phase column (GROM-SIL 100 ODS-2FE; GROM). Methanol/2-propanol (2 : 1, v/v) was used as the mobile phase and quinones were detected at 270 nm. DNA G+C content was determined by reversed-phase HPLC using the method of Tamaoka & Komagata (1984).
PCR amplification, sequencing and assembly of the 16S rRNA gene were carried out as described by Jeon et al. (2004). The resultant 16S rRNA gene sequence was compared with available 16S rRNA gene sequences from GenBank using the BLAST program to determine the approximate phylogenetic affiliation and was then aligned with those of alteromonas species and other related taxa using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were computed using SIMILARITY MATRIX version 1.1 (Ribosomal Database Project II; http://rdp.cme.msu.edu/html/analyses.html; Cole et al., 2003). Gaps at the 5'- and 3'-ends of the alignment were omitted from further analyses. Phylogenetic trees were constructed using three different methods, the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Evolutionary distance matrices were calculated according to the algorithm of the Kimura two-parameter model (Kimura, 1980) for the neighbour-joining method. To evaluate the stability of the phylogenetic tree, a bootstrap analysis (1000 replications) was performed with the SEQBOOT, DNADIST, NEIGHBOR and CONSENSE programs in the PHYLIP package.
Strain BH030046T on MA medium formed creamy and circular/slightly irregular colonies when grown at 35 °C for 2 days. The strain grew optimally on 2–5 % (w/v) NaCl-containing media and did not grow without NaCl or in the presence of more than 15 % (w/v) NaCl. Growth occurred at pH 6·5–9·0 (optimum 7·0–8·0) in 3 % (w/v) NaCl-containing nutrient broth. Growth was observed at temperatures of 10–45 °C (optimum 30–35 °C). Strain BH030046T was a Gram-negative, short rod, 0·8–1·0 µm in width and 1·2–1·5 µm in length. The strain was a strictly aerobic chemoheterotroph and showed obvious oxidase- and catalase-positive reactions, as reported for other closely related members of recognized species (Table 1). Spore formation was not observed and cells were motile by a single polar flagellum (see Supplementary Fig. A in IJSEM Online). Van Trappen et al. (2004) have reported that Alteromonas macleodii, Alteromonas marina and Alteromonas stellipolaris produce buds and prosthecae, but neither was observed on strain BH030046T.
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-D-lactose, L-rhamnose, glycerol, adonitol, D-raffinose, arbutin, D-salicin, D-melibiose or D-mannose. Using the API ZYM system, the strain showed activity of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Valine arylamidase and -chymotrypsin activities were weak and there was no lipase (C14), cystine arylamidase, trypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase or -fucosidase activity. Typical phenotypic and chemotaxonomic properties of strain BH030046T are summarized and compared with those of phylogenetically related type strains of the genus Alteromonas and the family Alteromonadaceae in Table 1
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The predominant isoprenoid quinone of strain BH030046T was ubiquinone-8 (Q-8) and the DNA G+C content was 48 mol%. The cellular fatty acid profiles of strain BH030046T and members of recognized Alteromonas species are given in Supplementary Table A in IJSEM Online. The predominant cellular fatty acids were straight-chain and unsaturated hydroxy fatty acids, such as C16 : 0, C18 : 17c and a mixture of C16 : 17c/iso-C15 : 0 2-OH, similar to the corresponding data for other Alteromonas species (Svetashev et al., 1995; Yoon et al., 2003, 2004). However, there were differences in the profiles of minor fatty acids, such as C16 : 0 N alcohol, C16 : 17c alcohol and C17 : 0 10-methyl, between the isolate and other members of the genus Alteromonas. Other related alteromonads produce C16 : 17c alcohol and C17 : 0 10-methyl, although only in small amounts, but these peaks were not detected from strain BH030046T.
An almost complete 16S rRNA gene sequence (1399 nt) of strain BH030046T was obtained and used for initial BLAST searches in GenBank and the phylogenetic analyses. Similarity values of strain BH030046 T to closely related recognized species were: Alteromonas macleodii DSM6062T (94·8 %), Alteromonas stellipolaris LMG 21861T (94·6 %), Alteromonas litorea KCCM 41775T (94·5 %), Alteromonas marina KCCM 41638T (94·1 %), Aestuariibacter halophilus JC2043T (93·5 %), Aestuariibacter salexigens JC2042T (92·1 %), Glaciecola punicea ACAM 611T (91·8 %), Glaciecola pallidula ACAM 615T (91·5 %) and Glaciecola mesophila KMM 241T (91·5 %). Phylogenetic analysis indicated that the isolate belonged to the -Proteobacteria. Strain BH030046T formed a distinct phyletic line with 100 % bootstrap value from the genera Alteromonas, Glaciecola and Aestuariibacter in the neighbour-joining analysis (Fig. 1). Maximum-likelihood (Supplementary Fig. B) and maximum-parsimony (data not shown) analyses also showed that the strain formed a phyletic clade distinguishable from the genus Alteromonas.
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Description of Salinimonas gen. nov.
Salinimonas (Sa.li.ni.mo'nas. L. fem. pl. n. salinae salterns, salt-works; L. fem. n. monas unit, monad; N.L. fem. n. Salinimonas monad from salterns).
Cells are Gram-negative, non-spore-forming short rods. Oxidase- and catalase-positive. Strictly aerobic, chemoheterotrophic and moderate halophilic. Cells are motile with a polar flagellum. No growth occurs without NaCl or in the presence of more than 15 % (w/v) NaCl. Major isoprenoid quinone is Q-8. DNA G+C content is 48 mol% (HPLC). Predominant cellular fatty acids are C16 : 0, C18 : 17c and a mixture of C16 : 17c/iso-C15 : 0 2-OH. Phylogenetically, the genus belongs to the family Alteromonadaceae.
The type species is Salinimonas chungwhensis.
Description of Salinimonas chungwhensis sp. nov.
Salinimonas chungwhensis (chung.when'sis. N.L. fem. adj. chungwhensis belonging to Chungwha, where the type strain was isolated).
Cells are approximately 0·8–1·0 µm wide and 1·2–1·5 µm long. Colonies are creamy and circular/slightly irregular on MA. Optimal growth is observed at 30–35 °C, pH 7–8 and 2–5 % (w/v) NaCl. Aesculin, casein, hypoxanthine, gelatin, starch, Tween 80, L-tyrosine and urea are hydrolysed. Hydrolysis of xanthine is not observed. Nitrate is not reduced to nitrite. Acid is produced from D-glucose, D-ribose, D-xylose, maltose, D-trehalose and L-arabinose, but not from D-fructose, D-mannitol, -D-lactose, L-rhamnose, glycerol, adonitol, D-raffinose, arbutin, D-salicin, D-melibiose and D-mannose. Produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase, but not lipase (C14), cystine arylamidase, trypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Valine arylamidase and -chymotrypsin activities are weak. Major isoprenoid quinone is Q-8. DNA G+C content is 48 mol% (HPLC).
The type strain, BH030046T (=KCTC 12239T=DSM 16280T), was isolated from a solar saltern in the Chungwha area in Korea.
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ACKNOWLEDGEMENTS |
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