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Ornithinimicrobium kibberense sp. nov., isolated from the Indian Himalayas

¹ Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh, 160 036, India
² Department of Microbiology, Guru Nanak Dev University (GNDU), Amritsar, 144 005, India

Correspondence
S. Mayilraj
mayil{at}imtech.res.in

	ABSTRACT

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A buff-yellow-pigmented bacterium, strain K22-20^T, which was isolated from a cold desert of the Indian Himalayas, was subjected to a polyphasic taxonomic study. Phenotypic and chemical properties of strain K22-20^T were consistent with its classification in the genus Ornithinimicrobium. The major fatty acids of the strain were iso-C_{17 : 1}9c (cis-15-methyl 7-hexadecenoic acid), iso-C_{15 : 0} (13-methyl tetradecanoic acid), iso-C_{16 : 0} (14-methyl pentadecanoic acid) and iso-C_{17 : 0} (15-methyl hexadecanoic acid). The G+C content of the genomic DNA was 71 mol%. According to 16S rRNA gene sequence analysis, strain K22-20^T was closely related to Ornithinimicrobium humiphilum HKI 0124^T (97.7 %). However, genomic relatedness between strain K22-20^T and O. humiphilum MTCC 6406^T, as revealed by DNA–DNA hybridization, was 64.5 %. Based on the polyphasic data, strain K22-20^T (=MTCC 6545^T=DSM 17687^T=JCM 12763^T) represents a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium kibberense sp. nov. is proposed.

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The suborder Micrococcineae Stackebrandt et al. 1997 currently consists of the following genera, which contain ornithine as diagnostic amino acid in their peptidoglycan: Cellulomonas Bergey et al. 1923, Curtobacterium Yamada and Komagata 1972, Rarobacter Yamamoto et al. 1988, Microbacterium Orla-Jensen 1919 emend. Takeuchi and Hatano 1998, Ornithinicoccus Groth et al. 1999, Salana von Wintzingerode et al. 2001, Ornithinimicrobium Groth et al. 2001, Rhodoglobus Sheridan et al. 2003, Arsenicicoccus Collins et al. 2004 and Serinicoccus Yi et al. 2004. At the time of writing, the genus Ornithinimicrobium consists of only one species, Ornithinimicrobium humiphilum. In this study, a novel strain is described, K22-20^T, which was isolated from a soil sample collected from Lahaul-Spiti valley in the Indian Himalayas.

Strain K22-20^T was isolated by the dilution-plate technique on tryptone soya agar medium (TSA; HiMedia) and maintained as glycerol stocks at –70 °C. The reference strain Ornithinimicrobium humiphilum MTCC 6406^T was taken from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. Strain K22-20^T and O. humiphilum MTCC 6406^T were grown on TSA at 28 °C and morphological and physiological properties were analysed. Colony and cell morphology were studied according to standard methods (Murray et al., 1994). Gram reaction was determined using the HiMedia Gram Staining kit according to the manufacturer's instructions. Physiological tests such as growth at different temperatures, pH and NaCl concentrations were examined by growing the strain on basal TSA. Catalase and urea hydrolysis were determined as described by Cowan & Steel (1965). Hydrolysis of casein, gelatin and starch, hydrogen sulphide production, methyl red and Voges–Proskauer tests and the presence of oxidase were determined as described by Smibert & Krieg (1994). Nitrate reduction and indole production were determined as described by Claus & Berkeley (1986). Utilization of various carbon sources was tested according to Reddy et al. (2003) and also using Biolog GP2 MicroPlates. The plates were used according to the manufacturer's instructions except that TSA medium was used instead of Biolog Universal Growth agar medium. Inoculated plates were incubated for 24 h and results were read with a MicroPlate Reader using Microlog 4.2 software. Acid production from various sugars was tested as described by Smith et al. (1952). Antibiotic susceptibility was tested by placing antibiotic discs (HiMedia) on Mueller–Hinton agar plates seeded with suspensions of strain K22-20^T.

Freeze-dried cells for chemotaxonomic analysis were prepared following growth of the strain in shake flasks containing tryptone soya broth (TSB; HiMedia) for 4 days at 28 °C. Whole-cell sugars were determined as described by Staneck & Roberts (1974). Peptidoglycan structure was determined using hydrolysates of purified cell walls according to Schleifer (1985). Amino acids and peptides were separated by two-dimensional ascending TLC as described by Schleifer & Kandler (1972) with the modification that TLC on cellulose sheets (Merck 5577) was used instead of paper chromatography. Phospholipids and menaquinones were extracted and analysed as described by Minnikin et al. (1984). The glycolic acid content of the bacterial cell wall was determined by the colour reaction method of Uchida et al. (1999). For cellular fatty acid analysis, cells were grown on TSA at 28 °C for 36 h and fatty acid methyl ester analysis was performed with the Sherlock Microbial Identification System (MIDI) as described previously (Pandey et al., 2002). DNA–DNA hybridization was performed by the membrane filter method (Tourova & Antonov, 1987). The genomic DNA G+C content was determined spectrophotometrically (Lambda35; Perkin Elmer) using the thermal denaturation method (Mandel & Marmur, 1968).

The chromosomal DNA of strain K22-20^T was isolated according to Rainey et al. (1996). The 16S rRNA gene was amplified with primers 8-27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1500r (5'-AGAAAGGAGGTGATCCAGCCA-3'). The amplified DNA fragment was separated on 1 % agarose gel, eluted from the gel and purified using a QIAquick gel extraction kit (Qiagen). The purified PCR product was sequenced with four forward and three reverse primers, namely 8-27f, 357f (5'-CTCCTACGGGAGGCAGCAG-3'), 704f (5'-TAGCGGTGAAATGCGTAGA-3'), 1114f (5'-GCAACGAGCGCAACC-3'), 685r (5'-TCTACGCATTTCACCGCTAC-3'), 1110r (5'-GGGTTGCGCTCGTTG-3') and 1500r (Escherichia coli numbering system). The 16S rRNA gene sequence was determined by the dideoxy chain-termination method with the Big-Dye terminator kit using an ABI 310 Genetic Analyzer (Applied Biosystems). The 16S rRNA gene sequence of strain K22-20^T generated in this work (1488 bases) was aligned with 16S rRNA gene sequences of O. humiphilum and other members of the family Intrasporangiaceae and suborder Micrococcineae. A sequence similarity search was done using GenBank BLASTN (Altschul et al., 1997). Sequences of closely related taxa were retrieved and aligned using the program CLUSTAL_X (Thompson et al., 1997) and the alignment was corrected manually. For neighbour-joining analysis (Saitou & Nei, 1987), distances between the sequences were calculated using Kimura's two-parameter model (Kimura, 1980). Bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein, 1985).

Detailed test reactions are presented in the species description. Based on biochemical characteristics, strain K22-20^T differed from the closely related species O. humiphilum (Table 1). Most of the chemotaxonomic properties (presented in the species description) were typical of members of the genus Ornithinimicrobium. Cellular fatty acids differed quantitatively between strain K22-20^T and the type strain of O. humiphilum (Table 2). To determine the phylogenetic relationships of strain K22-20^T, the 16S rRNA gene sequence (1488 bp) was compared with those of type strains of species of the genera retrieved from GenBank. Results revealed that strain K22-20^T is a member of the suborder Micrococcineae (Stackebrandt et al., 1997) (Fig. 1). Strain K22-20^T showed 97.7 % 16S rRNA gene sequence similarity to O. humiphilum HKI 0124^T and low similarity values (95.0–89.0 %) to members of the genera Ornithinicoccus, Demetria, Curtobacterium, Terracoccus, Promicromonospora, Beutenbergia, Kocuria, Dermatophilus, Sanguibacter, Cellulomonas, Stomatococcus, Arthrobacter, Bogoriella, Brachybacterium, Nesterenkonia, Rarobacter, Dermabacter, Microbacterium and Jonesia.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Neighbour-joining tree based on16S rRNA gene sequences (1488 bases) showing the phylogenetic relationship between strain K22-20T and Ornithinimicrobium humiphilum and other members of the family Intrasporangiaceae and families of the suborder Micrococcineae. Bootstrap values greater than 500 (1000 replications) are given at the nodes. Bar, 1 % sequence variation.

Description of Ornithinimicrobium kibberense sp. nov.
Ornithinimicrobium kibberense (kib.be.ren'se. N.L. neut. adj. kibberense pertaining to Kibber, a village in the Indian Himalayas, where the type strain was isolated).

Colonies on TSA are buff-yellow-pigmented and about 1–3 mm in diameter, circular, smooth, glistening, opaque and convex with an entire margin. Catalase-positive and oxidase-negative. Grows at 20–37 °C; optimum growth temperature is 28 °C and no growth occurs at 42 °C. NaCl is tolerated up to 7 %. Positive for hydrolysis of starch and nitrate reduction and variable for casein hydrolysis. Negative for indole, hydrogen sulphide production and methyl red and Voges–Proskauer tests. Acid is produced from D-fructose, D-galactose, raffinose, sucrose, trehalose and D-maltose, but not from salicin, D-glucose, D-melibiose, L-arabinose, D-mannose, inulin, D-lactose, L-rhamnose, D-xylose, cellobiose or D-mannitol. Utilization of various substrates as sole carbon sources and sensitivity to antibiotics are given in Table 1. The diagnostic cell wall amino acid is L-ornithine and the interpeptide bridge consists of L-Orn, L-Ala, Gly and D-Asp. Whole-cell sugars are glucose, arabinose and xylose. The acyl type is acetyl. Major fatty acids are iso-C_{17 : 1}9c (20.39 %), iso-C_{15 : 0} (19.42 %), iso-C_{16 : 0} (18.60 %) and iso-C_{17 : 0} (12.40 %). The phospholipid is of type V, with phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and unknown glucosamine-containing phospholipids. The major menaquinone is MK-8(H₄). Mycolic acids are absent. The DNA G+C content of the type strain is 71 mol%.

The type strain is K22-20^T (=MTCC 6545^T=DSM 17687^T=JCM 12763^T), isolated from soil, 45 cm below an ice glacier, 4200 m above sea level, in Kibber village of the Spiti valley, Himachal Pradesh, India.

	ACKNOWLEDGEMENTS

 
We thank Dr J. P. Euzéby, Ecole Nationale Veterinaire, France, for his suggestion on Latin nomenclature for the novel species. We would like to thank Mr Malkit Singh for his excellent technical assistance. P. S. is the recipient of the CSIR research fellowship. Financial assistance from the CSIR and DBT, Government of India is duly acknowledged. This is IMTECH communication number 50/2005.

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