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1 Department of Science Education, Cheju National University, Jeju 690-756, Republic of Korea
2 Department of Life Science, Cheju National University, Jeju 690-756, Republic of Korea
Correspondence
Soon Dong Lee
sdlee{at}cheju.ac.kr
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ABSTRACT |
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A table detailing the cellular fatty acid content of strain SSW1-57T and related species of the genus Aeromicrobium is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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to accommodate an aerobic, Gram-positive, motile actinomycete that showed coccoid or rod-shaped morphology; its description was emended recently by Yoon et al. (2005). Currently, the genus comprises four species, Aeromicrobium alkaliterrae (Yoon et al., 2005), Aeromicrobium erythreum (Miller et al., 1991), the type species of the genus, Aeromicrobium fastidiosum (Tamura & Yokota, 1994) and Aeromicrobium marinum (Bruns et al., 2003), which were isolated from environmental samples such as soils and seawater. Comparative studies of 16S rRNA gene sequences showed that the genus Aeromicrobium was phylogenetically related to members of the genera Marmoricola and Nocardioides within the radiation of the family Nocardioidaceae (Stackebrandt et al., 1997; Urzì et al., 2000; Yoon et al., 2005). In this paper, we describe the taxonomic status of a novel Aeromicrobium strain that was isolated from dried seaweed collected from a beach. During an investigation of the genetic diversity of polysaccharide-producing marine bacteria, strain SSW1-57T was isolated from dried seaweed collected from Samyang beach in Jeju Island, Korea, and subjected to morphological, cultural, physiological and chemotaxonomic characterization, in addition to 16S rRNA gene sequence studies. A dried seaweed sample (1 g) was placed into a sterile plastic tube containing 9 ml sterile distilled water. After mixing for 30 min using a tube rotator, aliquots (100 µl) of serial dilutions of the sample were transferred onto plates of isolation medium (SC-SW agar) containing 10 g soluble starch, 0.3 g casein, 2 g KNO3, 2 g NaCl, 0.02 g CaCO3, 0.05 g MgSO4.7H2O, 0.01 g FeSO4.7H2O and 18 g agar in a mixture of 600 ml natural seawater and 400 ml distilled water (pH 7.2). After incubation at 30 °C for 14 days, a colony from one plate was subcultivated on YE-SW agar (4 g yeast extract, 10 g malt extract, 4 g glucose and 18 g agar in a mixture of 600 ml natural seawater and 400 ml distilled water; pH 7.2). The pure culture was maintained at –20 and –80 °C in 20 % glycerol supplemented with 60 % (v/v) natural seawater.
Chromosomal DNA was extracted and purified by using a Wizard Genomic DNA Purification kit (Promega), according to the manufacturer's instructions. PCR amplification and sequencing of the 16S rRNA gene were performed as described previously (Lee et al., 2000). The CLUSTAL_X program (Thompson et al., 1997) was used to align an almost-complete 16S rRNA gene sequence of strain SSW1-57T (1406 nt; determined in this study) with corresponding sequences of members of the family Nocardioidaceae and related genera. Phylogenetic analyses were carried out using several tree-making algorithms contained in the PHYLIP package (Felsenstein, 1993). Bootstrap analysis (Felsenstein, 1985) was performed to evaluate the reliability of the tree topology. A total of 1333 unambiguous, aligned positions present in all strains between positions 59 and 1431 (Escherichia coli numbering; Brosius et al., 1978) were used for the final tree construction. A neighbour-joining tree based on 16S rRNA gene sequences (Fig. 1) showed that strain SSW1-57T belongs to the family Nocardioidaceae and formed a peripheral branch in the Aeromicrobium cluster, supported by a bootstrap value of 100 %. In trees constructed using maximum-parsimony and maximum-likelihood methods, strain SSW1-57T occupied an intermediate position between the A. alkaliterrae–A. marinum and A. erythreum–A. fastidiosum branches. The levels of 16S rRNA gene sequence similarity between strain SSW1-57T and its phylogenetic relatives were 96.7 % (with A. erythreum) and 96.5 % (with A. alkaliterrae, A. fastidiosum and A. marinum). Sequence similarities between strain SSW1-57T and other members of the family Nocardioidaceae were less than 94.2 %.
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). The DNA G+C content of strain SSW1-57T was 72.7 mol%. The isolate contained LL-diaminopimelic acid as the diagnostic diamino acid in the cell wall [wall chemotype I sensu Lechevalier & Lechevalier (1970)]. The predominant isoprenoid quinone was tetrahydrogenated menaquinone with nine isoprene units [MK-9(H4)], indicating that strain SSW1-57T contained a type of menaquinone that was different from that found in its phylogenetic neighbours Marmoricola and Nocardioides (Park et al., 1999; Urzì et al., 2000). Mycolic acids were not present. The phospholipid profile comprised phosphatidylinositol, diphosphatidylglycerol and phosphatidylglycerol [phospholipid type I pattern as defined by Lechevalier et al. (1981)]. Preparation and analysis of the cellular fatty acid methyl esters were performed using the Sherlock Microbial Identification System (version 6.0; MIDI), according to the instructions of the manufacturer. Strain SSW1-57T was grown for 3 days at 30 °C on trypticase soy broth (Difco) supplemented with Bacto agar (Difco). The cellular fatty acid profile of strain SSW1-57T was characterized by the presence (
1.0 %) of C18 : 1
9c (47.2 %), C16 : 0 (13.8 %), C18 : 0 (13.6 %) 10-methyl C18 : 0 (8.7 %), C16 : 0 2-OH (6.8 %), C17 : 0 (1.8 %), C16 : 19c (1.6 %), C12 : 0 (1.4 %), C17 : 0 2-OH (1.1 %), C14 : 0 (1.0 %) and C15 : 0 (1.0 %) acids. The major fatty acids were consistent with those of members of the genus Aeromicrobium (Park et al., 1999; Bruns et al., 2003; Yoon et al., 2005), as shown in Supplementary Table S1 in IJSEM Online. Thus, the chemotaxonomic markers, except for the phospholipid composition, were in agreement with the phylogenetic clustering of the novel isolate with the genus Aeromicrobium. The difference in polar lipids between the isolate and A. erythreum and A. fastidiosum, which contained phosphatidylethanolamine (Tamura & Yokota, 1994), suggests that the presence/absence of phospholipids with amino groups could be one phenotypic characteristic that differentiates the novel isolate from the two species. To date, the phospholipid compositions of A. alkaliterrae and A. marinum have not been reported.
Morphological and physiological characteristics were investigated using YE-SW agar at 30 °C. Cell morphology and motility were observed using phase-contrast and transmission electron microscopy with 3-day-old cultures. Colony pigmentation was observed visually and recorded using 5-day-old cultures on YE-SW agar at 30 °C. The Gram-reaction was determined by using a Gram-stain kit (bioMérieux), following the instructions of the manufacturer. Oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine was used to determine oxidase activity. Catalase activity was determined using 3 % (v/v) H2O2 solution. Growth at a range of temperatures (4–45 °C) was tested using YE-SW agar. Growth in various NaCl concentrations was studied using ISP 2 medium (Shirling & Gottlieb, 1966). NaCl was added to the basal medium at final concentrations of 0–9, 10 and 15 % (w/v). The pH range for growth was determined on YE-SW agar adjusted to pH 4.1–12.1 (at intervals of 1.0 pH units). The pH of the medium was adjusted prior to sterilization by addition of 6 M HCl or 10 M NaOH. Decomposition of hypoxanthine, DL-tyrosine and xanthine was examined as described by Gordon et al. (1974). Hydrolysis of casein, gelatin and starch was tested as described by MacFaddin (1980). The ability to utilize a variety of substrates as sole carbon and energy sources was tested using ISP 9 medium (Shirling & Gottlieb, 1966), with each filter-sterilized carbon source being used at a final concentration of 1 % (w/v) for carbohydrates and alcohols and 0.1 % (w/v) for organic acids. Strain SSW1-57T was grown on YE-SW agar at 30 °C for 3 days and the cells were then scraped from the agar surface with a cotton swab. The cells were washed and resuspended in sterile distilled water before being used. Other physiological and biochemical properties were tested using API 20E and API ZYM systems (bioMérieux), according to the instructions of the manufacturer. Morphological, physiological and biochemical properties are given in Table 1 and the species description.
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Description of Aeromicrobium tamlense sp. nov.
Aeromicrobium tamlense (tam.len'se. N.L. neut. adj. tamlense of Tamla, the old name of Jeju, Republic of Korea, the site of isolation of the type strain).
Aerobic, oxidase-negative and catalase-positive. Cells are Gram-positive irregular rods ranging in size from 0.4–0.6x0.8–1.2 to 0.5x3.8–4.8 µm. Non-motile. Branching or mycelial form does not occur. Endospores are not formed. Colonies are circular, smooth, convex, yellow and 0.6–0.8 mm in diameter after incubation for 5 days on YE-SW agar at 30 °C. Temperature range for growth is 10–42 °C, with optimum growth at 30 °C. No growth occurs at 4 or 45 °C. Growth occurs at pH 5.1–10.1, with optimum growth at pH 7.1. Growth occurs in the presence of 0–5 % NaCl. Nitrate is not reduced to nitrite. Gelatin liquefaction is observed. Aesculin is hydrolysed, but not casein. H2S and indole are not produced. Hypoxanthine is decomposed, but not DL-tyrosine or xanthine. Urease activity is not detected. In API 20E tests, the following enzymic activities are not detected: arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase. Voges–Proskauer reaction is weakly positive. Acetate, D-cellobiose, dextran, D-fructose, D-galactose, D-glucose, D-lactose, malate, maltose, D-mannose, methyl 
-D-glucoside, L-ribose, succinate, sucrose, D-trehalose and glycerol are utilized as sole carbon and energy sources. The following carbon sources are not utilized: D-arabinose, L-arabinose, benzoate, citrate, formate, inulin, D-melezitose, methyl -D-mannoside, D-raffinose, L-rhamnose, salicin, L-sorbose, tartrate, D-xylose, adonitol, 2,3-butanediol, D-dulcitol, meso-erythritol, myo-inositol, D-mannitol, 1,2-propanediol, D-sorbitol and D-xylitol. In API ZYM tests, alkaline phosphatase, leucine arylamidase, acid phosphatase and -glucosidase are positive. Esterase (C4), lipase (C14), valine arylamidase, cystine arylamidase, -chymotrypsin, -galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase are negative. The following are weakly positive: esterase lipase (C8), trypsin and naphthol-AS-BI-phosphohydrolase. Predominant cellular fatty acids are C18 : 19c, C16 : 0, 10-methyl C18 : 0, C18 : 0 and C16 : 0 2-OH. Polar lipids comprise phosphatidylinositol, diphosphatidylglycerol and phosphatidylglycerol.
The G+C content of the DNA of the type strain is 72.7 mol%. The type strain is strain SSW1-57T (=JCM 13811T=NRRL B-24466T), which was isolated from dried seaweed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bruns, A., Philipp, H., Cypionka, H. & Brinkhoff, T. (2003). Aeromicrobium marinum sp. nov., an abundant pelagic bacterium isolated from the German Wadden Sea. Int J Syst Evol Microbiol 53, 1917–1923.
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Park, Y.-H., Yoon, J.-H., Shin, Y. K., Suzuki, K.-i., Kudo, T., Seino, A., Kim, H.-J., Lee, J.-S. & Lee, S. T. (1999). Classification of ‘Nocardioides fulvus’ IFO 14399 and Nocardioides sp. ATCC 39419 in Kribbella gen. nov., as Kribbella flavida sp. nov. and Kribbella sandramycini sp. nov. Int J Syst Bacteriol 49, 743–752.
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Tamura, T. & Yokota, A. (1994). Transfer of Nocardioides fastidiosa Collins and Stackebrandt 1989 to the genus Aeromicrobium as Aeromicrobium fastidiosum comb. nov. Int J Syst Bacteriol 44, 608–611.
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Urzì, C., Salamone, P., Schumann, P. & Stackebrandt, E. (2000). Marmoricola aurantiacus gen. nov., sp. nov., a coccoid member of the family Nocardioidaceae isolated from a marble statue. Int J Syst Evol Microbiol 50, 529–536.[Abstract]
Yoon, J.-H., Lee, C.-H. & Oh, T.-K. (2005). Aeromicrobium alkaliterrae sp. nov., isolated from an alkaline soil, and emended description of the genus Aeromicrobium. Int J Syst Evol Microbiol 55, 2171–2175.
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