SGM Journals
Actinobacteria

Nocardioides mesophilus sp. nov., isolated from soil

Syed G. Dastager, Jae-Chan Lee, Ashok Pandey, Chang-Jin Kim

  • 1National Institute for Interdisciplinary Science and Technology (CSIR), Thiruvananthpuram-695019, India
  • 2Korea Research Institute of Bioscience and Biotechnology 52, Oeun-dong, Yuseong-gu, Daejeon, 305-333, Republic of Korea
  • Correspondence
    Chang-Jin Kim
    changjin{at}kribb.re.kr

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(10):2288–2292 · https://doi.org/10.1099/ijs.0.019059-0

    View at publisher PubMed

    Abstract

    A short coccoid- to rod-shaped, motile, mesophilic actinobacterium, strain MSL-22T, was isolated from soil on Bigeum Island, Korea. A polyphasic study was undertaken to establish the taxonomic position of this strain. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MSL-22T formed an evolutionary lineage within the radiation of the genus Nocardioides. In particular, it formed a monophyletic lineage with Nocardioides jensenii KCTC 9134T with which it shared the highest sequence similarity of about 97.3%. However, DNA–DNA relatedness demonstrated that strain MSL-22T was distinct from its closest phylogenetic neighbours. The cell-wall peptidoglycan of strain MSL-22T contained ll-diaminopimelic acid. The predominant menaquinone was MK-8(H4). Strain MSL-22T had a cellular fatty acid profile containing straight-chain, branched, unsaturated and 10-methyl fatty acids, with iso-C16 : 0 as the major fatty acid. The DNA G+C content of the strain was 68.7 mol%. On the basis of phenotypic and phylogenetic evidence, the strain is separate from previously described members of the genus Nocardioides and represents a novel species in this genus, for which the name Nocardioides mesophilus sp. nov. is proposed. The type strain is MSL-22T (=DSM 19432T=KCTC 19310T).

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain MSL-22T is EF466117.

    • A scanning electron micrograph of cells of strain MSL-22T is available with the online version of this paper.

    The genus Nocardioides was proposed by Prauser (1976) for nocardioform actinomycetes that form mycelia which fragment into irregular rod- or coccus-like elements. The organisms are Gram-positive, non-acid-fast, aerobic and mesophilic, and have a cell wall based on ll-diaminopimelic acid, the diagnostic diamino acid of cell-wall chemotype I. At the time of writing, the genus Nocardioides is composed of over 40 species with validly published names, including the recently described species N. bigeumensis, N. agariphilus, N. koreensis, N. sediminis, N. halotolerans, N. tritolerans, N. islandensis, N. dilutus (Dastager et al., 2008a, b, c, d, e, 2009), N. salarius (Kim et al., 2008), N. hwasunensis (Lee et al., 2008), N. hankookensis (Yoon et al., 2008), N. fonticola (Chou et al., 2008), N. dokdonensis (Park et al., 2008) and N. daphniae (Tóth et al., 2008). The aim of the present study was to determine the taxonomic position of strain MSL-22T using a polyphasic taxonomic approach.

    Strain MSL-22T was isolated after 7 days at 28 °C on 10 % R2A agar (Reasoner & Geldreich, 1985) from a soil sample collected from Bigeum Island (Jeollanam-do Province, Korea) and prepared using the standard dilution technique. To investigate its morphological, physiological and biochemical characteristics, strain MSL-22T was routinely cultivated at 28 °C on 50 % R2A agar and preserved in glycerol suspensions (20 %, v/v) at –80 °C. Gram-staining was performed using a Gram-stain kit (bioMérieux), according to the manufacturer's instructions. Cell morphology and motility were studied using phase-contrast microscopy and scanning electron microscopy (Philips SEM 515; JEOL).

    The physiological characteristics of strain MSL-22T were examined on 50 % R2A medium. Growth at various temperatures (4–50 °C) was measured on 50 % R2A agar. Growth at different pH values (pH 6.0–12.0) was determined by adjusting the pH with Na2CO3 prior to sterilization and incubating for 5 days. Growth at various NaCl concentrations from 0–20 % (w/v) was investigated on 50 % R2A agar at 28 °C for 5 days. Catalase and oxidase activities and hydrolysis of starch, casein, gelatin, hypoxanthine, Tween 80, l-tyrosine, urea and xanthine were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin and nitrate reduction were studied as described by Lányí (1987). Enzyme activity was determined by using the API ZYM system (bioMérieux). Utilization of various substrates as sole carbon and energy sources was determined as described by Shirling & Gottlieb (1966).

    Cell biomass for DNA extraction and analysis of cell components was harvested from 50 % R2A broth after incubation at 28 °C for 7 days. The G+C content of the genomic DNA of strain MSL-22T was determined using HPLC with a reversed-phase column (GROM-SIL 100 ODS-2FE; GROM), according to the method of Tamaoka & Komagata (1984). The cell wall was purified and the amino acids of the peptidoglycan were analysed by TLC (Lechevalier & Lechevalier, 1980). Analysis of the whole-cell sugar composition followed procedures described by Becker et al. (1965). Phospholipid analysis was carried out as described by Lechevalier et al. (1977). Menaquinones were determined using the procedures of Collins et al. (1977). For fatty acid methyl ester analysis, cell mass of strain MSL-22T was harvested from 50 % R2A plates after incubation at 28 °C for 7 days. The fatty acids were extracted, methylated and analysed using the standard Microbial Identification System (MIDI; Sasser, 1990; Kämpfer & Kroppenstedt, 1996). The G+C content of the genomic DNA of strain MSL-22T was 68.7 mol%. The major respiratory lipoquinone of strain MSL-22T was MK-8(H4). The fatty acid profile of strain MSL-22T showed a predominance of saturated fatty acids: iso-C16 : 0 (44.6 %), C16 : 1 (12.1 %), iso-C14 : 0 (11.0 %), C16 : 0 (6.5 %), C18 : 1ω9c (3.1 %), 10-methyl-C16 : 0 (2.8 %), iso-C15 : 0 (2.3 %) and C15 : 0 (2.2 %). Similar fatty acid profiles have been reported for type strains of species of the genus Nocardioides.

    Strain MSL-22T formed cream–white, glistening, translucent, slightly sticky, irregular and slightly raised colonies when grown on 50 % R2A agar at 28 °C for 3 days. Optimum growth was observed at 28 °C (range 20–37 °C) and at pH 7.0–7.5. Cells of strain MSL-22T were Gram-positive, motile, short irregular rods, 0.3–0.8 μm wide and 0.9–1.4 μm long (see Supplementary Fig. S1, available in IJSEM Online) with no flagella. Strain MSL-22T was oxidase-negative and catalase-positive and did not reduce nitrate to nitrite. Growth was not observed after 5 days at 28 °C on 50 % R2A agar under anaerobic conditions.

    The typical phenotypic characteristics of strain MSL-22T are summarized and compared with those of the type strains of closely related taxa in Table 1⇓ and additional characteristics are given in the species description. Strain MSL-22T exhibited characteristics of the genus Nocardioides (iso-C16 : 0 as the major fatty acid and DNA G+C contents in the range of 67–74 mol%; Prauser, 1976, 1984; Yoon & Park, 2006).

    Table 1.

    Differential phenotypic properties of strain MSL-22T and related strains of the genus Nocardioides

    Strains: 1, MSL-22T; 2, N. jensenii DSM 20641T; 3, N. dubius KCTC 9992T; 4, N. alkalitolerans KCTC 19037T; 5, N. dilutus DSM 19318T; 6, N. daphniae DSM 18664T. Data from identical conditions were obtained from this study, Collins et al. (1994), Yoon et al. (1997, 2004), Dastager et al. (2008c) and Tóth et al. (2008). All strains were rods or cocci and positive for Gram-stain, casein, d-cellobiose, d-glucose, sucrose and d-xylose utilization and acid phosphatase, esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase. All strains were negative for l-arabinose, d-ribose, maltose, hypoxanthine and xanthine utilization and urease, α-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. +, Positive; v, variable; w, weakly positive; −, negative; nd, no data available.

    Genomic DNA extraction and PCR amplification of the 16S rRNA gene of strain MSL-22T were carried out using the procedure of Chun & Goodfellow (1995). The 16S rRNA gene sequence of strain MSL-22T was compared with sequences available in GenBank by using blast () to determine the approximate phylogenetic affiliation and was aligned with close relatives by using clustal w (Thompson et al., 1994). An almost-complete 16S rRNA gene sequence (1403 nt) of strain MSL-22T was obtained and a neighbour-joining (Saitou & Nei, 1987) phylogenetic analysis showed that strain MSL-22T was most closely affiliated to the genus Nocardioides (Fig. 1⇓). Strain MSL-22T exhibited the highest 16S rRNA gene sequence similarity (97.3 %) to Nocardioides jensenii KCTC 9134T and formed a distinct monophyletic cluster with this type strain. Lower 16S rRNA gene sequence similarities were found with the type strains of other species of the genus Nocardioides (93.8–96.6 %) and the other taxa included in the phylogenetic analysis (<94.7 %). The topologies of the phylogenetic trees generated with the maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) algorithms also showed that strain MSL-22T belonged to the genus Nocardioides (data not shown). Because of the high pairwise 16S rRNA gene sequence similarity between strain MSL-22T and N. jensenii KCTC 9134T, DNA–DNA hybridization was performed using the optical renaturation method (De Ley et al., 1970; Huß et al., 1983; Jahnke, 1992) in duplicate under optimal hybridization conditions. DNA–DNA relatedness between strain MSL-22T and N. jensenii DSM 20641T was 34±2.0 %, which is well below the 70 % threshold value recommended for the definition of bacterial species (Wayne et al., 1987).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining dendrogram based on 16S rRNA gene sequences indicating the phylogenetic position of strain MSL-22T. Bootstrap values (>50 %) based on 1000 resamplings are shown at branch nodes. Terrabacter tumescens KCTC 9133T served as an outgroup. Bar, 1% sequence divergence.

    Thus, on the basis of physiological, biochemical and phylogenetic properties, strain MSL-22T represents a novel species within the genus Nocardioides, for which the name Nocardioides mesophilus sp. nov. is proposed.

    Description of Nocardioides mesophilus sp. nov.

    Nocardioides mesophilus [me.so.phi′lus. Gr. adj. mesos middle; Gr. adj. philos loving; N.L. masc. adj. mesophilus middle (temperature)-loving, mesophilic].

    Gram-positive, motile without flagella, aerobic, non-spore-forming, short irregular rods (0.3–0.8×0.9–1.4 μm). Colonies are cream–white, glistening, translucent, slightly sticky, irregular and slightly raised with entire margins after 72 h at 28 °C on 50 % R2A agar. Optimum growth occurs at 28 °C and pH 7.0–7.5. Catalase-positive and oxidase-negative. Casein, starch and tyrosine are hydrolysed, but aesculin, gelatin, Tween 80 and urea are not. Nitrate is not reduced to nitrite. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. Negative for lipase (C14), cystine arylamidase, valine arylamidase, trypsin, α-chymotrypsin, α- and β-galactosidase, β-glucuronidase, α- and β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. As sole carbon and energy sources, utilizes cellobiose, glucose, inositol, mannitol, raffinose, sucrose and xylose, but not aesculin, fructose, galactose, lactose, maltose, mannose, melibiose, rhamnose or ribose. Acid is not produced from any of the carbon sources tested. The main cell-wall amino acid is ll-diaminopimelic acid. The predominant menaquinone is MK-8(H4). The cellular fatty acids are iso-C16 : 0, C16 : 1, iso-C14 : 0, C16 : 0, C18 : 1ω9c, 10-methyl-C16 : 0, iso-C15 : 0 and C15 : 0.

    The type strain, MSL-22T (=DSM 19432T=KCTC 19310T), was isolated from a soil sample collected from Bigeum Island in South Korea. The DNA G+C content of the type strain is 68.7 mol%.

    Acknowledgments

    This work was supported by the 21C Frontier Program of Microbial Genomics and Applications from the Korean Ministry of Education, Science & Technology (MEST) and by a grant from KRIBB Research Initiative Program, Korea.

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