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Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, South Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
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ABSTRACT |
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7c and/oriso-C15 : 0 2-OH. The DNA G+C contents of the two isolates were 59.8 and 60.0 mol%. Isolates UMS-37T and UMS-40 exhibited no difference in their 16S rRNA gene sequences and possessed a mean DNA–DNA relatedness level of 94 %; they exhibited 16S rRNA gene sequence similarity levels of 96.8–98.2 % to the type strains of recognized Herbaspirillum species. Phylogenetic analyses based on 16S rRNA gene sequences showed that isolates UMS-37T and UMS-40 formed a distinct phylogenetic lineage within the genus Herbaspirillum. DNA–DNA relatedness levels between isolates UMS-37T and UMS-40 and the type strains of some phylogenetically related Herbaspirillum species were in the range 3–56 %. On the basis of differences in phenotypic properties and phylogenetic distinctiveness and genomic data, isolates UMS-37T and UMS-40 were classified in the genus Herbaspirillum within a novel species, for which the name Herbaspirillum rhizosphaerae sp. nov. is proposed, with the type strain UMS-37T (=KCTC 12558T =CIP 108917T).
Detailed DNA–DNA hybridization results are available as supplementary material with the online version of this paper.
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; Valverde et al., 2003). The genus accommodates bacteria that are Gram-negative, motile by means of one to three or more flagella, generally vibrioid and sometimes helical in shape (Baldani et al., 1986, 1996; Ding & Yokota, 2004; Im et al., 2004). The genus Herbaspirillum is also characterized chemotaxonomically by having Q-8 as the predominant ubiquinone and by DNA G+C contents of 57.9–67 mol% (Baldani et al., 1986, 1996; Kirchhof et al., 2001; Valverde et al., 2003; Ding & Yokota, 2004; Im et al., 2004; Rothballer et al., 2006). At present, the genus consists of nine species with validly published names: H. seropedicae (Baldani et al., 1986), Herbaspirillum rubrisubalbicans (Baldani et al., 1996), H. frisingense (Kirchhof et al., 2001), H. lusitanum (Valverde et al., 2003), H. chlorophenolicum (Im et al., 2004), H. autotrophicum (Aragno & Schlegel, 1978; Ding & Yokota, 2004), H. huttiense (Leifson, 1962; Ding & Yokota, 2004), H. putei (Ding & Yokota, 2004) and H. hiltneri (Rothballer et al., 2006). In this study, we report on the detailed taxonomic characterization of two Gram-negative, milky-white-pigmented, slightly curved rod-shaped bacterial isolates, UMS-37T and UMS-40, which were isolated from the rhizosphere soil of Allium victorialis var. platyphyllum, wild edible greens cultivated on Ulleung island, Korea.
Isolates UMS-37T and UMS-40 were isolated from the same rhizosphere soil sample of Allium victorialis var. platyphyllum using the standard dilution plating technique at 30 °C on nutrient agar (NA; Difco). To investigate their morphological, physiological and biochemical characteristics, the isolates were routinely cultivated at 30 °C on NA. Growth at various temperatures from 4 to 40 °C was measured on NA and tolerance to various NaCl concentrations was measured in nutrient broth (NB; Difco). Optimal pH and pH range for growth were determined in NB that was adjusted to various pH values (pH 4.5–10.5 at intervals of 0.5 pH units). Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on NA and on NA supplemented with nitrate, both of which had been prepared anaerobically using nitrogen. The cell morphology and presence of flagella were examined by light microscopy (Nikon E600) and transmission electron microscopy by using cells grown on NA. The Gram reaction was determined by using the bioMérieux Gram stain kit according to the manufacturer's instructions. Catalase and oxidase activities and hydrolysis of casein, starch, hypoxanthine, xanthine, tyrosine and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were studied as described by Lanyi (1987). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates for growth was determined as described by Yurkov et al. (1994). The API ZYM system (bioMérieux) was used to determine enzyme activity. Antibiotic sensitivity was tested by spreading bacterial suspension on NA and applying discs impregnated with the following antibiotics (content per disc); ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), chloramphenicol (100 µg), gentamicin (30 µg), lincomycin (15 µg), kanamycin (30 µg), neomycin (30 µg), novobiocin (5 µg), oleandomycin (15 µg), penicillin G (20 U), polymyxin B (100 U), streptomycin (50 µg) and tetracycline (30 µg).
Isolates UMS-37T and UMS-40 were cultivated for 3 days in NB at 30 °C to obtain the cell mass required for isoprenoid quinone analysis and DNA extraction. Isoprenoid quinones were analysed as described previously (Komagata & Suzuki, 1987), using reversed-phase HPLC. For fatty acid methyl ester (FAME) analysis, cell mass of isolates UMS-37T and UMS-40 was harvested from NA plates after cultivation for 3 days at 30 °C. The FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). Chromosomal DNA was extracted and purified by a procedure described previously (Yoon et al., 1996). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984) with the modification that DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. DNA–DNA reassociation was determined by the microplate hybridization method (Ezaki et al., 1989) using photobiotin-labelled DNA probes. Type strains of eight Herbaspirillum species were used as reference strains for DNA–DNA hybridization: H. autotrophicum DSM 732T, H. huttiense DSM 10281T and H. frisingense DSM 13128T were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany; H. chlorophenolicum KCTC 12096T was obtained from the Korean Collection for Type Cultures (KCTC), Taejon, Korea, and H. hiltneri LMG 23131T, H. lusitanum LMG 21710T, H. rubrisubalbicans LMG 2286T and H. seropedicae LMG 6513T were obtained from the BCCM/LMG Bacteria Collection, Ghent University, Ghent, Belgium. 16S rRNA gene amplification was performed according to the method described previously using two universal primers (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003). PCR primers and conditions used to detect the nifD and nifH genes were described by Stoltzfus et al. (1997), Poly et al. (2001) and Im et al. (2004).
Morphological, cultural, physiological and biochemical characteristics of isolates UMS-37T and UMS-40 are shown in Table 1 or are given in the species description. The isolates did not grow on semi-solid, nitrogen-free JNFb medium (Döbereiner, 1995), and nifD and nifH genes were not detected by PCR-based methods, indicating that the two isolates do not possess the ability to fix atmospheric nitrogen. The 16S rRNA gene sequences of isolates UMS-37T and UMS-40 determined in this study comprised 1488 nucleotides and were identical. Comparative 16S rRNA gene sequence analysis and estimation of phylogenetic relationships showed that isolates UMS-37T and UMS-40 are phylogenetically most closely related to the genus Herbaspirillum (Fig. 1). They exhibited 16S rRNA gene sequence similarity of 96.8–98.2 % to the type strains of Herbaspirillum species. Sequence similarities to other species included in the phylogenetic analysis were less than 95.7 %. Isolates UMS-37T and UMS-40 exhibited a mean DNA–DNA relatedness value of 94 % when their DNAs were used individually as labelled DNA probes for cross-hybridization. This value indicated that isolates UMS-37T and UMS-40 are members of the same genomic species (Wayne et al., 1987). Levels of DNA–DNA relatedness between isolates UMS-37T and UMS-40 and the type strains of eight Herbaspirillum species that showed 16S rRNA gene sequence similarity values of >97.0 % to the two isolates were in the range 3–56 % (DNA–DNA relatedness levels are detailed in Supplementary Table S1 available in IJSEM Online).
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; Ding & Yokota, 2004). The cellular fatty acid profiles of isolates UMS-37T and UMS-40 are shown in Table 2, together with those of some type strains of Herbaspirillum species. The fatty acid profiles were characterized by large amounts of straight-chain, unsaturated, hydroxy and cyclopropane fatty acids; the major components (>10 % of total fatty acids) were C16 : 0, cyclo C17 : 0 and C16 : 17c and/or iso-C15 : 0 2-OH. The fatty acid profiles of two isolates were similar to those of Herbaspirillum species, although there were differences in the proportions of some fatty acids, which might be caused by different cultivation or analytical conditions (Table 2). The DNA G+C contents of isolates UMS-37T and UMS-40 were 60.0 and 59.8 mol%, respectively. The differential phenotypic properties, together with the phylogenetic distinctiveness and DNA–DNA relatedness data, provide decisive evidence that isolates UMS-37T and UMS-40 differ from recognized Herbaspirillum species (Table 1
). On the basis of the data presented, isolates UMS-37T and UMS-40 should be classified as members of a novel species of the genus Herbaspirillum, for which the name Herbaspirillum rhizosphaerae sp. nov. is proposed.
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Cells are Gram-negative, slightly curved rods, 0.3–0.4x1.8–2.2 µm. Motile by means of bipolar flagella. No growth occurs under anaerobic conditions on NA or on NA with nitrate. Growth occurs at 4 and 34 °C, with optimum growth at 25–30 °C. Optimal pH for growth is 6.5–7.5; growth occurs weakly at pH 5.0 but not at pH 4.5. No growth occurs in the presence of more than 3 % (w/v) NaCl. Hypoxanthine, tyrosine and urea are hydrolysed, but aesculin, casein, gelatin, starch, xanthine and Tweens 20 and 60 are not. Acetate, citrate, glycerol, pyruvate, ribose and succinate are utilized as sole carbon and energy sources. D-Xylose is utilized, but adonitol, benzoate, D-cellobiose, formate, L-glutamate, maltose, salicin, sucrose and trehalose are not utilized. Utilization of L-malate (positive for type strain) and L-arabinose (negative for type strain) is variable. Acid is produced from D-glucose, D-galactose, D-mannitol, D-mannose, D-ribose, D-xylose and L-arabinose. Acid is not produced from D-cellobiose, lactose, myo-inositol, maltose, melibiose, D-melezitose, D-sorbitol, sucrose, L-rhamnose or D-raffinose. Acid production from D-fructose (positive for type strain) and trehalose (negative for type strain) is variable. Susceptible to chloramphenicol, gentamicin, kanamycin, neomycin, oleandomycin, streptomycin and tetracycline but not to ampicillin, carbenicillin, cephalothin, lincomycin, novobiocin, penicillin G or polymyxin B. The major cellular fatty acids (>10 % of total fatty acids) are C16 : 0, cyclo C17 : 0 and C16 : 17c and/or iso-C15 : 0 2-OH. The predominant ubiquinone is Q-8. The DNA G+C content of the type strain is 60.0 mol%.
The type strain, UMS-37T (=KCTC 12558T =CIP 108917T), was isolated from rhizosphere soil of Allium victorialis var. platyphyllum, wild edible greens cultivated on Ulleung island, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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