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1 Biochemical Engineering Research and Process Development Centre (BERPDC) and Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh 160 036, India
2 Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
Correspondence
S. Mayilraj
mayil{at}imtech.res.in
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain RMV-1378T is AY426714.
Chemotaxonomic characteristics of strain RMV-1378T and the type strain of A. cyanogriseus are available in a supplementary table in IJSEM Online.
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but the name of the genus was not validly published. Subsequently, Itoh et al. (1987) reported the morphological and chemotaxonomic characteristics of the genus, and its phylogenetic position was reported by Tamura & Hatano (1998) and Stackebrandt et al. (1997). The genus name was finally validly published by Tamura et al. (2000). At present, this genus accommodates only one species, Actinoalloteichus cyanogriseus. In this paper, we describe the polyphasic characterization of strain RMV-1378T and propose it as the type strain of a novel species. Strain RMV-1378T was isolated from a soil sample collected from the Lahaul–Spiti Valley, a cold desert of the Indian Himalayas, by using a dilution plating technique on actinomycetes isolation agar (sodium caseinate, 0·2 %; asparagine, 0·01 %; sodium propionate, 0·4 %; dipotassium phosphate, 0·05 %; magnesium sulphate, 0·01 %; ferrous sulphate, 0·0001 %; agar, 1·5 %; pH 8·1) and maintained as glycerol stocks at –70 °C. The type strain of A. cyanogriseus (MTCC 6195T) was obtained from the Microbial Type Culture Collection, Chandigarh, India.
Cultural, physiological and biochemical characteristics (Table 1) of strain RMV-1378T were examined by using standard procedures (Yokota et al., 1993; Gordon et al., 1974). Some tests were re-examined in this study as indicated in Table 1. To check growth at different pH values between 8·0 to 11·0, CM broth (yeast extract, 0·4 %; malt extract, 1·0 %; glucose, 0·4 %) with different pH values along with a biological buffer system were used (a solution of Na2CO3, using 25 % as stock, was used for pH adjustment). Freeze-dried cells for chemotaxonomic analyses were prepared after growth of the strains for 4 days at 30 °C in shake flasks containing trypticase soy broth. The whole-cell sugars were analysed by using the HPLC method of Mikami & Ishida (1983) and the diaminopimelic acid isomer was determined as described by Staneck & Roberts (1974). Phospholipids and menaquinones were extracted and analysed as described by Minnikin et al. (1984). The absence of mycolic acids was confirmed by TLC (Minnikin & Goodfellow, 1976). The N-acyl type of the muramic acid in the cell wall was determined by using the method of Uchida & Aida (1984). Fatty acid methyl ester analysis was performed by using GLC according to the instructions of the Microbial Identification System (MIDI) (Sasser, 1990).
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). DNA–DNA hybridization was performed by using the membrane filter method (Tourova & Antonov, 1987) as described by Reddy et al. (2000). The G+C content of the genomic DNA was determined using the HPLC method as described by Tamaoka & Komagata (1984).
The 16S rRNA gene sequence of strain RMV-1378T generated in this work (1476 bases) was aligned, using the CLUSTAL_X program (Thompson et al., 1997), with the 16S rRNA gene sequences of closely related strains (retrieved from the GenBank/EMBL/DDBJ databases); the alignment was corrected manually. A sequence-similarity search was done using the BLASTN program of GenBank (Altschul et al., 1997). For the neighbour-joining analysis (Saitou & Nei, 1987), distances between sequences were calculated using Kimura's two-parameter model (Kimura, 1980). A bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein, 1985).
Strain RMV-1378T showed a high level of 16S rRNA gene sequence similarity (99·3 %) to the sole member of the genus Actinoalloteichus, A. cyanogriseus (Fig. 1); sequence similarities to all other species (with validly published names) of related genera were below 95 %. The DNA–DNA hybridization experiments revealed 51·9 % relatedness (the mean of 50·1 and 53·9 %) between RMV-1378T and the type strain of A. cyanogriseus, which is well below the 70 % value recommended for the delineation of bacterial species (Wayne et al., 1987). Strain RMV-1378T exhibited good growth on Streptomyces agar (malt extract, 1·0 %; yeast extract, 0·4 %; glucose, 0·4 %; calcium carbonate, 0·2 %; agar, 1·2 %). Neither aerial mycelium nor spore formation was observed on the media tested (ISP media 1, 2, 3, 4, 5, 6, 7 and 9, Streptomyces agar and actinomycetes isolation agar). No soluble pigment was observed on the above media. Greyish colonies developed on all media tested. Detailed phenotypic characteristics are presented in the species description. Strain RMV-1378T differs substantially from A. cyanogriseus in terms of phenotypic characteristics (Table 1
). Most of the chemotaxonomic data confirmed that strain RMV-1378T belongs to the genus Actinoalloteichus (see Supplementary Table S1 available in IJSEM Online). The cell-wall peptidoglycan contained meso-diaminopimelic acid as a diagnostic amino acid. The whole-cell sugars comprised rhamnose, ribose, mannose, galactose and glucose. The acyl type of the glycan chain of peptidoglycan was acetyl. The major menaquinone was MK-9(H4) (82 %); MK-10(H4) (9 %), MK-8(H4) (5 %) and MK-9(H6) (2 %) were also detected. The predominant cellular fatty acids were iso-branched C16 : 0 (33 %) and C15 : 0 (17 %) fatty acids and anteiso-branched C15 : 0 (7 %) and C17 : 0 (8 %) fatty acids. The phospholipid profile consisted of phosphatidylmonoethylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylinositolmannosides and diphosphatidylglycerol.
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Description of Actinoalloteichus spitiensis sp. nov.
Actinoalloteichus spitiensis (spi.ti.en'sis. N.L. masc. adj. spitiensis pertaining to Spiti Valley, located in the Indian Himalayas, where the type strain was isolated).
Gram-positive, non-acid-fast, aerobic actinobacterium with branching, non-fragmenting vegetative hyphae. No aerial mycelium or spores produced. Positive for utilization of D-mannitol, D-raffinose, sucrose, salicin, sodium citrate and sodium succinate as sole carbon sources. Negative for utilization of D-glucose, D-maltose, myo-inositol, D-fructose, D-arabinose, D-xylose, L-rhamnose, D-mannose and D-sorbitol as sole carbon sources. Positive for decomposition of casein and negative for decomposition of urea. No growth occurs on MacConkey agar or in Sabouraud dextrose broth. Positive for hydrolysis of starch and negative for hydrolysis of hippurate and aesculin. Tolerates up to 2 % NaCl and grows at temperatures between 20 and 37 °C, with an optimum temperature of 25 °C; cannot grow at 15 or 42 °C. Growth occurs at initial pH values between 6 and 11, the optimum being pH 8·0. Chemotaxonomic characteristics correspond to those typical for the genus Actinoalloteichus. Contains major amounts of iso-branched C16 : 0 (33·0 %) and C15 : 0 (17·0 %) fatty acids and anteiso-branched C15 : 0 (7·0 %) and C17 : 0 (8·0 %) fatty acids. Contains a large amount of MK-9(H4) (82 %) and does not contain MK-9(H2). The G+C content of the DNA is 72·0 mol%.
The type strain, RMV-1378T (=MTCC 6194T=JCM 12472T=DSM 44848T), was isolated from soil 3600 m above sea level, at Rangrik Village in Spiti Valley, Himachal Pradesh, India.
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ACKNOWLEDGEMENTS |
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