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Division of Biology and Ocean Sciences, Inha University, Incheon 402-751, Republic of Korea
Correspondence
Jang-Cheon Cho
chojc{at}inha.ac.kr
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ABSTRACT |
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-hydroxybutyrate granules. Based on 16S rRNA gene sequence comparisons, the most closely related species to strain IMCC1728T was Methylibium petroleiphilum (97.0 % similarity). Phylogenetic trees generated using 16S rRNA gene sequences showed that this isolate formed an independent phyletic line of the genus Methylibium clade of the class Betaproteobacteria. The DNA G+C content of the strain was 66.2±0.4 mol%. The predominant cellular fatty acid constituents were C16 : 1
7c and/or iso-C15 : 0 2-OH (43.1 %), C16 : 0 (20.3 %), C12 : 0 (13.4 %) and C10 : 0 3-OH (7.3 %). The strain contained Q-8 as the predominant ubiquinone. Several phenotypic characteristics, including flagellation, temperature range for growth and carbon source utilization patterns, differentiated strain IMCC1728T from species of the genus Methylibium. Therefore, it is proposed that strain IMCC1728T represents a novel species, Methylibium aquaticum sp. nov. The type strain is IMCC1728T (=KCCM 42364T=NBRC 102349T).
-hydroxybutyrate; TEM, transmission electron microscopyThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain IMCC1728T is DQ664244.
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) in the class Betaproteobacteria was recently established by Nakatsu et al. (2006) and the genus currently contains one species, Methylibium petroleiphilum. M. petroleiphilum was isolated from a compost biofilter. This species is a Gram-negative, rod-shaped, facultative methylotrophic bacterium that is motile with one polar flagellum and can degrade methyl tert-butyl ether. Characterization of strain IMCC1728T, a novel species of the genus Methylibium isolated from a eutrophic freshwater pond, is described in this report.
Strain IMCC1728T was isolated from an artificial freshwater pond located inside Inha University (Incheon, Korea) by using a standard dilution plating method on R2A agar (Reasoner & Geldreich, 1985) diluted in distilled water (1 : 10, v/v) after aerobic incubation at 20 °C for 5 days. After determination of optimum medium and temperature for growth, bacterial cultures were routinely maintained on R2A agar at 30 °C.
Methods for DNA extraction, PCR and 16S rRNA gene sequencing have been described in a previous study (Cho & Giovannoni, 2003). The resultant almost-complete 16S rRNA gene sequence (1484 bp) of strain IMCC1728T was aligned with sequences of nearest neighbours using the ARB software package (Ludwig et al., 2004) and 16S rRNA gene sequence similarities were calculated based on this alignment. Unambiguously aligned 1294 nt positions, determined from 13 betaproteobacterial 16S rRNA gene sequences, were used for phylogenetic analyses in PAUP* 4.0 beta 10 (Swofford, 2002). Phylogenetic trees were generated by neighbour-joining (Saitou & Nei, 1987) with the Jukes–Cantor distance (Jukes & Cantor, 1969), maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. Robustness of the neighbour-joining and maximum-parsimony trees was confirmed by bootstrap analyses based on 100 resamplings of the sequences. Preliminary sequence comparison with 16S rRNA gene sequences deposited in GenBank and RDP-II (Cole et al., 2005) indicated that strain IMCC1728T was closely related to members of the genus Methylibium in the class Betaproteobacteria. Strain IMCC1728T showed highest 16S rRNA gene sequence similarity to M. petroleiphilum ATCC BAA-1232T (97.0 %), followed by Ideonella dechloratans ATCC 51718T (96.1 %), Aquabacterium commune ATCC BAA-209T (95.7 %) and Aquabacterium parvum ATCC BAA-208T (95.6 %). In all phylogenetic trees generated in this study (Fig. 1), strain IMCC1728 T, uncultured groundwater bacterium 300A-D08 (GenBank accession no. AY662010; Fields et al., 2005) and uncultured iron-oxidizing lagoon bacterium 015 (GenBank accession no. AB252910) formed a monophyletic clade. This clade was anchored to the clade containing M. petroleiphilum with high bootstrap values (92 % in neighbour-joining and 83 % in maximum-parsimony trees), indicating that strain IMCC1728T represents a novel species of the genus Methylibium.
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-hydroxybutyrate (PHB) granules was confirmed using the Nile Blue A staining method described by Ostle & Holt (1982). Colony morphology, size and colour were examined from cultures grown for 3 days. Motility was tested by using wet mounts of exponential-phase cells. Anaerobic growth was tested employing Anaerocult C mini (EM science) and AnaeroPack Anaero (Mitsubishi Gas Chemical). Catalase activity was determined by the reaction of 3 % hydrogen peroxide on fresh colonies and the oxidase test was performed using Kovacs' solution (Kovacs, 1956). Other biochemical tests were carried out by using the API 20NE and API ZYM (both bioMérieux) systems according to the manufacturer's instructions. Sole carbon source utilization tests were performed using custom-made 48-well microplates. Approximately 1.4x104 cells ml–1 in PBS (pH 7.4) were inoculated into each well, which contained one of 47 different carbon compounds (list of compounds is given in the species description) at a final concentration of 0.2 % (w/v or v/v). After incubation of the microplates for 1 week at 30 °C, cellular growth and purity were checked as described previously (Cho & Giovannoni, 2003). Susceptibility to 10 different antimicrobial agents (list given in the species description) was determined using the diffusion plate method (Jorgensen et al., 1999). The DNA G+C content was analysed by using HPLC according to Mesbah et al. (1989) with a Discovery C18 column (5 µm, 15 cmx4.6 mm; Supelco). Cellular fatty acid methyl esters were collected from fresh cultures grown on R2A agar at 30 °C for 4 days and analysed according to the instructions of the Microbial Identification System (MIDI) by the Korean Culture Center of Micro-organisms (KCCM). Respiratory quinone content was determined by using reverse-phase HPLC analysis (Komagata & Suzuki, 1987) by the KCCM.
In summary, cells of strain IMCC1728T were Gram-negative, chemoheterotrophic, facultatively aerobic, non-motile, short rods that contained PHB granules (Fig. 2). Detailed results of phenotypic and biochemical tests are given in the species description and Table 1. As shown in Table 1, strain IMCC1728T shared similar characteristics with M. petroleiphilum ATCC BAA-1232T, including DNA G+C content, major fatty acid constituents, Q-8 as the major quinone type and the presence of PHB granules. However, differences in several phenotypic properties (Table 1) together with a phylogenetically distinct relationship (Fig. 1
) between the two strains indicate that strain IMCC1728T represents a novel species of the genus Methylibium, for which the name Methylibium aquaticum sp. nov. is proposed.
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Gram-negative and non-motile. Cells are short rods (0.8–1.9x0.4–0.7 µm). After 4 days incubation, colonies on R2A agar are circular, umbonate, beige-milkish, smooth with entire margin and 0.5–2.0 mm in diameter. Growth occurs at 15–42 °C and pH 6–10 and in 0–1 % NaCl; optimal growth is observed at 30 °C, pH 7.0 and in the absence of NaCl. Contains PHB granules. Chemoheterotrophic and facultatively aerobic. Anaerobic growth is much slower than aerobic growth. Catalase-negative and oxidase-positive. Gelatinase activity and aesculin hydrolysis are weakly positive. Nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease and -galactosidase are negative (API 20NE). Using the API ZYM system, alkaline phosphatase and leucine arylamidase activities are positive; esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities are negative. Of the 47 different carbon sources in the custom-made 48-well plate assay, the following carbon substrates are utilized: methanol, glycerol, D-ribose, D-galactose, N-acetyl-D-glucosamine, rhamnose, adonitol, D-xylitol, gluconic acid, malonic acid, propionic acid, L-alanine, L-arginine, L-histidine, L-proline and L-serine. However, methylamine, ethanol, DL-glyceraldehyde, D-arabinose, D-xylose, D-fructose, D-glucosamine, D-glucose, D-mannose, D-cellobiose, D-lactose, maltose, melibiose, sucrose, trehalose, D-melezitose, D-raffinose, arabitol, D-mannitol, myo-inositol, D-sorbitol, citric acid, glucuronic acid, itaconic acid, pyruvic acid, succinic acid, L-glutamic acid, L-glycine, L-leucine, L-lysine and L-ornithine are not utilized. Susceptible to (µg) chloramphenicol (25), erythromycin (15), streptomycin (10), gentamicin (10), kanamycin (30), penicillin G (10) and vancomycin (30). Resistant to (µg) ampicillin (10), rifampicin (50) and tetracycline (30). The cellular fatty acids are composed of C16 : 17c and/or iso-C15 : 0 2-OH (43.1 %), C16 : 0 (20.3 %), C12 : 0 (13.4 %), C10 : 0 3-OH (7.3 %), cyclo C17 : 0 (4.2 %), C18 : 17c (1.5 %), C15 : 0 (1.4 %), C17 : 0 (1.4 %), C14 : 0 (1.0 %), C15 : 16c (0.9 %), C18 : 0 (0.8 %) and 11-methyl C18 : 17c (0.6 %). The major quinone type is Q-8. The DNA G+C content of the type strain is 66.2±0.4 mol%.
The type strain, IMCC1728T (=KCCM 42364T=NBRC 102349T), was isolated from an artificial freshwater pond located inside Inha University, Incheon, Korea.
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ACKNOWLEDGEMENTS |
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