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1 Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon 305-600, Korea
2 Department of Genetic Engineering, Paichai University, Taejon 302-735, Korea
Correspondence
Jung-Hoon Yoon
jhyoon{at}kribb.re.kr
Jung-Kee Lee
jklee{at}kribb.re.kr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A2-4T is DQ218275.
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; Swift et al., 1996; Fuqua & Greenberg, 1998). This type of signalling mechanism depends on small, diffusible signal molecules, N-acylhomoserine lactones (AHLs), all of which have an identical homoserine lactone ring, although the acyl side-chain length and substitutions in the side-chain differ. The quorum-sensing system plays an important role in the control of virulence-gene expression in pathogenic bacteria, including Erwinia carotovora (Jones et al., 1993) and Pseudomonas aeruginosa (Pearson et al., 1994, 1997). Enzymic degradation of AHL molecules was reported as a useful strategy for disrupting the quorum-sensing signal system, thereby interfering with infection in the case of many pathogenic bacteria. Some AHL-degrading micro-organisms and enzymes have been reported recently (Dong et al., 2000; Leadbetter & Greenberg, 2000; Lee et al., 2002; Flagan et al., 2003; Park et al., 2003; Uroz et al., 2003). In this study, an AHL-degrading Nocardioides-like bacterial strain, A2-4T, which was isolated from a soil sample, is described. To date, bacteria that are able to degrade AHLs have not been reported for the genus Nocardioides. Accordingly, the aim of the present study was to determine the exact taxonomic position of strain A2-4T by using a polyphasic taxonomic approach.
Soil samples collected from Kongju, Korea, were used as the source for the isolation of AHL-degrading bacteria. The soil samples (5 mg) were inoculated in 2 ml modified minimal medium (Leadbetter & Greenberg, 2000), adjusted to pH 6.5, containing N-hexanoyl-L-homoserine lactone (HHL) as the carbon source and NH4Cl as the nitrogen source. This medium was incubated at 30 °C on a horizontal shaker at 150 r.p.m. After incubation for 3 days, 0.1 ml suspension was transferred into 2 ml fresh medium and the medium was reincubated under the same conditions. After three successive transfers, the suspension was plated on medium solidified with 1.5 % (w/v) agar in order to isolate pure cultures. Of the isolates, strain A2-4T showed high levels of HHL-degrading activity and was selected for further study. The utilization of HHL as a sole carbon source was investigated in modified minimal medium (Leadbetter & Greenberg, 2000) containing 1 mM HHL. The amounts of residual HHL in the growth medium were measured as described by Park et al. (2003). Nocardioides simplex KCTC 9106T, Nocardioides nitrophenolicus KCTC 0457BPT and Nocardioides aromaticivorans JCM 11674T were used as reference strains for DNA–DNA hybridization and phenotypic characterization.
To investigate its morphological, physiological and biochemical characteristics, strain A2-4T was routinely cultivated at 30 °C on nutrient agar (Difco) and in nutrient broth (Difco). Morphological, physiological, cultural and biochemical properties were examined as described by Yoon et al. (2005a). Growth at various NaCl concentrations (0–8 %, w/v, in increments of 1.0 %) was investigated in trypticase soy broth prepared according to the formula of the Difco medium except that no NaCl was used. The pH range for growth was determined in nutrient broth adjusted to various pH values (pH 4.0–10.5, in increments of 0.5 pH units). The pH was adjusted, prior to sterilization, to various levels by the addition of HCl and Na2CO3. Other physiological properties were tested by using the API 20E system (bioMérieux). Cell biomass for DNA extraction and for analyses of the cell wall and the isoprenoid quinones was obtained by cultivation for 3 days in nutrient broth at 30 °C. For fatty acid methyl ester analysis, cell mass of strain A2-4T was harvested from nutrient agar after incubation for 6 days at 30 °C. Chemotaxonomic and molecular systematic studies were performed as described by Yoon et al. (2005a). The isomer type of the diamino acid in the cell-wall peptidoglycan was analysed using TLC according to the method described by Komagata & Suzuki (1987). DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the means of the remaining three values were quoted as the DNA–DNA relatedness values.
Strain A2-4T grew with HHL as the sole carbon source. As the cell density increased, the residual amount of HHL in the culture medium decreased (Fig. 1). Most of the HHL in the culture medium was consumed after cultivation for 10 h (Fig. 1). No growth was observed in an HHL-depleted minimal medium. The details of HHL degradation by strain A2-4T will be described elsewhere. The morphological, cultural, physiological and biochemical characteristics of strain A2-4T are given in the species description (see below) or are shown in Table 1, together with those of N. simplex and N. nitrophenolicus. The almost complete 16S rRNA gene sequence of strain A2-4T, comprising 1475 nt (approx. 96 % of the Escherichia coli 16S rRNA sequence), was determined in this study. Comparative 16S rRNA gene sequence analyses showed that strain A2-4T fell within the phylogenetic radiation encompassed by the genus Nocardioides, forming, in particular, a coherent cluster with N. nitrophenolicus, N. aromaticivorans and N. simplex (Fig. 2). In the neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, strain A2-4T clustered with N. nitrophenolicus with a high level of bootstrap confidence (91.2 %), and this cluster joined the phylogenetic lineage comprising N. simplex and N. aromaticivorans with a bootstrap resampling value of 100 % (Fig. 2). Strain A2-4T exhibited 16S rRNA gene sequence similarity values of 99.1, 98.6 and 98.3 % with respect to N. nitrophenolicus KCTC 0457BPT, N. aromaticivorans H-1T and N. simplex KCTC 9106T, respectively, and values of less than 96.3 % (the similarity with Nocardioides pyridinolyticus) with respect to the type strains of the other Nocardioides species. The values for sequence similarity with respect to other species used in the phylogenetic analysis were in the range 89.5–94.2 %.
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. The predominant isoprenoid quinone detected in strain A2-4T was a tetrahydrogenated menaquinone with eight isoprene units [MK-8(H4)]. The cellular fatty acids of strain A2-4T (i.e. those fatty acids constituting >1 % of total fatty acids) were as follows: branched fatty acids iso-C16 : 0 (27.1 %), iso-C17 : 0 (6.3 %), anteiso-C17 : 0 (3.0 %), iso-C15 : 0 (2.2 %), iso-C18 : 0 (1.4 %) and iso-C16 : 1 (1.3 %); unsaturated fatty acids C17 : 1
6c (11.2 %), C18 : 19c (5.0 %) and C17 : 18c (2.9 %); straight-chain fatty acids C16 : 0 (3.9 %), C18 : 0 (3.4 %) and C17 : 0 (1.4 %); 10-methyl fatty acids C18 : 0 (14.4 %), C17 : 0 (6.0 %) and C16 : 0 (3.2 %); summed feature 3, comprising C16 : 17c and/or iso-C15 : 0 2-OH (2.3 %); and hydroxy fatty acid C16 : 0 2-OH (1.5 %). This fatty acid profile was similar to those of Nocardioides species, particularly those of N. simplex and N. nitrophenolicus in that the major fatty acids (i.e. those which each constituted >10 % of total fatty acids) are iso-C16 : 0, 10-methyl C18 : 0 and C17 : 16c, although there are differences in the proportions of some fatty acids, perhaps because of differences in extraction and cultivation conditions (Yoon et al., 1997, 1999, 2004, 2005a, b; Lawson et al., 2000; Yi & Chun, 2004a, b; Kubota et al., 2005). The DNA G+C content of strain A2-4T was 72.1 mol%. These chemotaxonomic properties were in agreement with the result of the phylogenetic analysis, showing the affiliation of strain A2-4T with the genus Nocardioides (Tamura & Yokota, 1994; Yoon et al., 1997; Lawson et al., 2000; Urzì et al., 2000; Wang et al., 2001).
The levels of DNA–DNA relatedness between strain A2-4T and the type strains of three phylogenetically related Nocardioides species, namely N. nitrophenolicus KCTC 0457BPT, N. simplex KCTC 9106T and N. aromaticivorans JCM 11674T, were 38, 28 and 22 %, respectively, when their DNAs were used individually as labelled DNA probes. The corresponding relative binding values for the reciprocal experiment in which the DNA of strain A2-4T was labelled were 46, 21 and 36 %, respectively. Strain A2-4T was differentiated from its closest phylogenetic neighbours N. simplex, N. nitrophenolicus and N. aromaticivorans by the phenotypic properties listed in Table 1
. The phylogenetic distinctiveness of the novel strain, the differential phenotypic properties and the DNA–DNA relatedness data were sufficient to categorize strain A2-4T as a species that is distinct from previously recognized Nocardioides species (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Therefore, on the basis of the data presented, strain A2-4T should be classified in the genus Nocardioides as a member of a novel species, for which the name Nocardioides kongjuensis sp. nov. is proposed.
Description of Nocardioides kongjuensis sp. nov.
Nocardioides kongjuensis (kong.ju.en'sis. N.L. masc. adj. kongjuensis of Kongju, Korea, from where the type strain was isolated).
Cells are aerobic, non-spore-forming rods (0.4–0.7x0.8–3.0 µm) in the exponential phase of growth. Cells show rod-to-coccus morphogenesis from the early exponential phase to the stationary phase. Gram-positive. Colonies are irregular, smooth, flat, yellowish white in colour and 3.0–5.0 mm in diameter after 3 days incubation on nutrient agar at 30 °C. Neither substrate mycelium nor aerial mycelium is formed. Growth occurs at 10 and 40 °C, but not at 4 or 41 °C. Optimal pH for growth is 7.0–8.0; growth occurs at pH 5.5, but not at pH 5.0. Growth occurs in the presence of 0–5 % (w/v) NaCl; optimum growth occurs in the absence of NaCl. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase. H2S and indole are not produced. Tweens 20, 40 and 60 are hydrolysed. D-Trehalose, L-malate and pyruvate are utilized, but formate and salicin are not. The diagnostic diamino acid in the cell-wall peptidoglycan is LL-2,6-diaminopimelic acid. The predominant menaquinone is MK-8(H4). The major fatty acids (>10 % of total fatty acids) are iso-C16 : 0, 10-methyl C18 : 0 and C17 : 16c. The DNA G+C content is 72.1 mol% (determined by HPLC). Other phenotypic characteristics are given in Table 1.
The type strain, A2-4T (=KCTC 19054T=JCM 12609T), was isolated from a soil in Kongju, Korea.
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ACKNOWLEDGEMENTS |
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) and without HHL (
) was assessed. The residual HHL concentration (
) in the culture medium was measured by using a bioassay (Park et al., 2003
-glucosidase and utilization of D-glucose, sucrose and succinate. All taxa are negative for nitrate reduction*
, hydrolysis of xanthine*
-galactosidase, 