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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Environmental Biotechnology National Core Research Center, PMBBRC, Division of Environmental Biotechnology, Gyeongsang National University, 660-701, Republic of Korea
3 Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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8c. Strains M9T and M18T could grow in the presence of up to 13–15 % (w/v) NaCl, but their optimum salt concentrations were relatively low (0–3 %, w/v). The major predominant isoprenoid quinone was Q-8 and the G+C content of the genomic DNA was 57–58 mol%. Phylogenetic analyses and comparative 16S rRNA gene sequence studies revealed that strains M9T and M18T formed a phylogenetic lineage distinct from the genus Teredinibacter within the class Gammaproteobacteria and were most closely related to the genera Microbulbifer, Saccharophagus and Teredinibacter, with less than 92·5 % 16S rRNA gene sequence similarity. The level of 16S rRNA gene sequence similarity between the two strains was 96·7 %. On the basis of physiological and phylogenetic properties, strains M9T and M18T represent separate species within a novel genus of the class Gammaproteobacteria, for which the names Marinimicrobium koreense gen. nov., sp. nov. (type species) and Marinimicrobium agarilyticum sp. nov. are proposed. The type strains of Marinimicrobium koreense and Marinimicrobium agarilyticum are M9T (=KCTC 12356T=DSM 16974T) and M18T (=KCTC 12357T=DSM 16975T), respectively.
The GenBank accession numbers for the 16S rRNA gene sequences of strains M9T and M18T are AY839869 and AY839870.
An electron micrograph of a cell of strain M18T, a photograph of colonies showing agarolytic activity and details of other properties of the novel isolates are available as supplementary material in IJSEM Online.
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; Martín et al., 2003) and many novel species have recently been added to the genera (Gorshkova et al., 2003; Romanenko et al., 2005; Satomi et al., 1998, 2004; Shivaji et al., 2005; Yoon et al., 2004). For example, Microbulbifer hydrolyticus isolated from a lignin-rich pulp mill waste is capable of degrading lignin and lignin-like polymers and Marinobacter hydrocarbonoclasticus, with hydrocarbon-degrading activity, was isolated from an oil-producing well on an offshore platform in southern Vietnam (Huu et al., 1999; González et al., 1997; Gauthier et al., 1992; Marquez & Ventosa, 2005). In addition, Distel et al. (2002) isolated a moderate halophile, Teredinibacter turnerae T7902T, with cellulolytic activity from gill tissue of the shipworm. In this paper, we report the isolation of two novel moderately halotolerant, Gram-negative bacteria (strains M9T and M18T) from tidal flat sediment in Korea; strain M18T showed agar-dissolving activity. We determined their phylogenetic position using a polyphasic approach as members of two different species within a new genus of the class Gammaproteobacteria. Two strains, M9T and M18T, were isolated from a tidal flat of Jeonnam province in the South Sea of Korea (the Korea Strait). For isolation, sediment sample was collected from the surface of tidal flat sediment and diluted serially in saline solution (10 % w/v). The diluted soil samples were spread on marine agar 2216 (MA) (Difco) with the addition of 8 % (w/v) NaCl (final concentration, 9·94 % w/v NaCl) and incubated for 2 days at 30 °C. The isolates were routinely cultivated on MA for 2 days at 35 °C except where indicated otherwise. Requirement for and tolerance of NaCl were determined in trypticase soy broth (Difco) medium with different amounts of NaCl after 2 days incubation at 35 °C. Anaerobic growth was determined by incubation in an anaerobic chamber at 35 °C for 5 days on MA. Optimum growth was tested at different temperatures (4–55 °C) on MA and at different pH values (5·0–10·0) in marine broth.
Gram staining was determined using the bioMérieux Gram stain kit according to the manufacturer's instructions. Catalase activity was determined by production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide solution. Oxidase activity was tested by oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck). Nitrate reduction was assessed according to the method of Lanyi (1987). Hydrolysis of aesculin, casein, starch, Tween 20, Tween 80, hypoxanthine, tyrosine, gelatin, agar and xanthine was determined on MA according to the methods described by Cowan & Steel (1965), Lanyi (1987) and Gerhardt et al. (1994). The API ZYM system (bioMérieux) was used to determine the activities of some enzymes. Acid production from carbohydrates was tested as described by Leifson (1963); all suspension media were supplemented with artificial sea water containing 2 % (w/v) NaCl.
Cell morphology was studied using light microscopy and transmission electron microscopy. Motility was observed at 12 and 36 h on agar-coated wet mounts with a light microscope (Nikon E600). Agar-coated wet mounts for motility observations were prepared by placing 10 µl of a culture under a cover glass on a glass slide previously coated with a film of 0·5 % (w/v) agarose (Cambrex) in distilled water and air-dried. For investigation of flagellum type from the isolates, specimens were prepared from the exponential growth phase as described elsewhere (Lee et al., 2005) and then subjected to transmission electron microscopy (JEOL JEM-1010). Whole-cell fatty acids of strains M9T and M18T were analysed using GC/MS following the instructions of the Microbial Identification System (MIDI; Microbial ID, Inc.) after cultivation on MA for 2 days at 35 °C. Isoprenoid quinones and polar lipids were analysed as described by Komagata & Suzuki (1987). The DNA G+C content of strains M9T and M18T was determined by reversed-phase HPLC, using the method of Tamaoka & Komagata (1984).
The 16S rRNA gene sequences of strains M9T and M18T were analysed as described previously (DeLong, 1992). The resultant 16S rRNA gene sequences of strains M9T and M18T for phylogenetic analysis comprised 1454 and 1455 nucleotides, respectively, and were compared with available sequences from GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/) to determine the approximate phylogenetic affiliation. They were aligned with sequences from closely related organisms using the CLUSTAL W software (Thompson et al., 1994). Unmatched regions of the 5' and 3' ends from the alignment, which were caused by the different lengths of the 16S rRNA gene sequences, were deleted. Phylogenetic trees based on 16S rRNA gene sequences were constructed using three different methods, neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) algorithms available in the PHYLIP software, version 3.6 (Felsenstein, 2002). Using the FASTA3 program in EBI, 16S rRNA gene sequence comparisons for similarity calculations were made between the isolated strains and other related organisms. A bootstrap analysis was performed according to the algorithm of the Kimura two-parameter model (Kimura, 1980) of the neighbour-joining method in the PHYLIP package.
Cells of strains M9T and M18T were non-spore-forming, short rods, approximately 0·5–0·8x0·9–1·1 µm and 0·6–0·7x0·9–1·5 µm, respectively (Supplementary Fig. S1 available in IJSEM Online). They were aerobic, Gram-negative, catalase-negative and motile with a flagellum. Colonies of strains M9T and M18T were creamy, smooth, glistening, low-convex and circular/slightly irregular. Growth of strains M9T and M18T was not observed under anaerobic conditions. Strains M9T and M18T showed optimum growth at low NaCl concentrations of 1–3 and 0–1 % (w/v), respectively. However, they could grow in the presence of up to 13–15 % (w/v) NaCl, meaning that they are moderately halotolerant. Strains M9T and M18T grew in the range of pH 6·0–10·5 in marine broth; optimal growth was observed at pH 7·0–7·5 and 7·0–8·0, respectively. The isolates displayed mesophilic growth, with optimum growth at 35–40 °C.
The predominant isoprenoid quinone of strains M9T and M18T was ubiquinone-8 (Q-8). The genomic DNA G+C contents of strains M9T and M18T were respectively 57 and 58 mol%. Strains M9T and M18T contained C16 : 0, C19 : 0 cyclo 8c and summed feature 3 (C16 : 17c and/or iso C15 : 0 2-OH) as the major cellular fatty acids on MA, but their fatty acid profiles were slightly different (Table 1). Most cultural and biochemical characteristics of strains M9T and M18T were similar, but there were some differences. For example, cells of strain M9T showed an oxidase-negative reaction, but those of strain M18T were oxidase-positive. Strain M9T contained large amounts of phosphatidylethanolamine and diphosphatidylglycerol and small amounts of two unknown phospholipids (PL1, PL2) as the polar lipids, while strain M18T contained only a large amount of phosphatidylethanolamine and a small amount of diphosphatidylglycerol (data not shown). Strain M18T also hydrolysed agar, whereas strain M9T did not (Supplementary Fig. S2). In enzyme production, such as 
- and 
-galactosidase and -glucosidase activities, they also had a few different characteristics. Further physiological and biochemical characteristics of strains M9T and M18T are compared in detail in the species descriptions and in Supplementary Table S1. Other morphological, physiological and biochemical characteristics of strains M9T and M18T are compared with those of phylogenetically related type strains in Table 2.
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). The topologies of phylogenetic trees built using the ML and MP algorithms also supported the conclusion that strains M9T and M18T formed a phylogenetic line distinct from these genera (data not shown). Comparative 16S rRNA gene sequence analyses showed that strains M9T and M18T were most closely related to members of the genera Microbulbifer, Saccharophagus and Teredinibacter, with no more than 92·5 % 16S rRNA gene sequence similarity. The level of 16S rRNA gene sequence similarity between strains M9T and M18T was 96·7 %, which suggests that the strains should be classified within the same genus and different species (Rossello-Mora & Amann, 2001; Stackebrandt et al., 2002). Therefore, on the basis of physiological and molecular properties, strains M9T and M18T represent a novel genus, Marinimicrobium gen. nov., and two different species within the class Gammaproteobacteria, for which the names Marinimicrobium koreense gen. nov., sp. nov. and Marinimicrobium agarilyticum sp. nov. are proposed.
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Strictly aerobic, chemoheterotrophic and moderately halotolerant. Colonies are creamy, smooth, glistening and circular/slightly irregular. Cells are Gram-negative, non-spore-forming, short rods, approximately 0·5–0·8 µm wide and 0·9–1·5 µm long. Nitrate is not reduced to nitrite. Cells are motile by means of a flagellum. Catalase-negative. The predominant isoprenoid quinone is Q-8. The major fatty acids are C16 : 0, C19 : 0 cyclo 8c and summed feature 3 (C16 : 1 7c and/or iso C15 : 0 2-OH). The G+C content of the genomic DNA is 57–58 mol% (HPLC). Phylogenetically, the genus belongs to the class Gammaproteobacteria. The type species is Marinimicrobium koreense.
Description of Marinimicrobium koreense sp. nov.
Marinimicrobium koreense (ko.re.en'se. N.L. neut. adj. koreense pertaining to Korea).
Growth of cells occurs at salinities in the range 0–15 % (w/v) NaCl (optimum 1–3 % w/v). Oxidase-negative. Grows at between 10 and 45 °C (optimum 35–40 °C) and from pH 6·0 to 10·5 (optimum pH 7·0–7·5). API ZYM kit gives positive results for alkaline phosphatase, esterase (C4), esterase lipase, leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase and N-acetyl--glucosaminidase and negative results for lipase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, -galactosidase, -glucuronidase, -glucosidase, -mannosidase and -fucosidase. Aesculin, starch and Tween 20 are hydrolysed. Casein, gelatin, L-tyrosine, xanthine and Tween 80 are not hydrolysed. Acids are produced from D-glucose, D-fructose, D-ribose, D-xylose, -D-lactose, maltose, D-trehalose, L-arabinose, D-melibiose, D-mannose and sucrose, but not from D-mannitol, adonitol, raffinose, glycerol or inositol. Shows no agarolytic activity. Contains large amounts of phosphatidylethanolamine and diphosphatidylglycerol and small amounts of two unknown phospholipids (PL1, PL2) as the polar lipids. The DNA G+C content is 57 mol%.
The type strain is strain M9T (=KCTC 12356T=DSM 16974T), isolated from tidal flat sediment in Korea.
Description of Marinimicrobium agarilyticum sp. nov.
Marinimicrobium agarilyticum (a.ga.ri.ly'ti.cum. N.L. n. agarum agar; N.L. adj. lyticus from Gr. adj. lutikos dissolving; N.L. neut. adj. agarilyticum agar-dissolving).
Cells grow at salinities in the range 0–13 % (w/v) NaCl (optimum 0–1 % w/v). Oxidase-positive. Grows at between 12 and 40 °C (optimum 35 °C) and from pH 6·0 to 10·5 (optimum pH 7·0–8·0). API ZYM kit gives positive results for alkaline phosphatase, esterase (C4), esterase lipase, valine arylamidase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, -galactosidase and N-acetyl--glucosaminidase and negative results for lipase, cystine arylamidase, trypsin, -chymotrypsin, acid phosphatase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, -mannosidase and -fucosidase. Agar, aesculin, starch, Tween 80 and Tween 20 are hydrolysed. Casein, gelatin, L-tyrosine and xanthine are not hydrolysed. Acids are produced from D-glucose, D-fructose, D-ribose, D-xylose, -D-lactose, maltose, D-trehalose, L-arabinose, raffinose, rhamnose, D-melibiose, D-mannose and sucrose, but not from D-mannitol, adonitol, glycerol or inositol. Shows agarolytic activity. Contains a large amount of phosphatidylethanolamine and a small amount of diphosphatidylglycerol as the polar lipids. The DNA G+C content is 58 mol%.
The type strain is strain M18T (=KCTC 12357T=DSM 16975T), isolated from tidal flat sediment in Korea.
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ACKNOWLEDGEMENTS |
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