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Int J Syst Evol Microbiol 57 (2007), 2272-2274; DOI  10.1099/ijs.0.65056-0
© 2007 International Union of Microbiological Societies

Catenulispora rubra sp. nov., an acidophilic actinomycete isolated from forest soil

Tomohiko Tamura, Yuumi Ishida, Takeshi Sakane and Ken-ichiro Suzuki

NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan

Correspondence
Tomohiko Tamura
tamura-tomohiko{at}nite.go.jp


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In the course of screening novel secondary metabolites, an acidophilic actinomycete strain, designated Aac-30T, was isolated from forest soil and subjected to a polyphasic taxonomic characterization study. It grew well on media in which the pH ranged from 4 to 6, but not on medium with pH adjusted to 7. It possessed LL-diaminopimelic acid and glycine in the cell-wall peptidoglycan, MK-9(H6) and MK-9(H8) as major isoprenoid quinones, iso-C16 : 0 and anteiso-C17 : 0 as major cellular fatty acids, and phosphatidylglycerol and phosphatidylinositol as polar lipids. The G+C content of the genomic DNA was 69.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that the organism belonged to the family Catenulisporaceae and consistently formed a monophyletic cluster with members of the genus Catenulispora. Physiological, biochemical and chemotaxonomic data revealed that this novel organism could be readily differentiated from recognized members of the genus Catenulispora and that it merits separate species status. Based on the phenotypic and genetic evidence presented, strain Aac-30T is considered to represent a novel species of the genus Catenulispora, for which the name Catenulispora rubra sp. nov. is proposed. The type strain is Aac-30T (=NBRC 101179T=DSM 44948T).

Abbreviations: LL-A2pm, LL-diaminopimelic acid

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Aac-30T is AB180773.

A table detailing the cellular fatty acid profiles of strain Aac-30T and Catenulispora acidiphila is available with the online version of this paper.


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The genus Catenulispora was proposed by Busti et al. (2006)Go to accommodate a group of actinomycetes containing LL-diaminopimelic acid (LL-A2pm) and glycine in the cell-wall peptidoglycan. The type, and only, species of the genus, Catenulispora acidiphila, is reported to be acidophilic. In the course of isolating acidophilic micro-organisms for screening bioactive substances, strain Aac-30T was recovered from a forest soil sample collected at Oodate, Akita Prefecture, Japan, on an acidic medium for isolation. The isolation medium was prepared by mixing separately prepared solutions A (4 g glucose, 2 g Difco yeast extract and 18 g agar in 500 ml distilled water; pH adjusted to 5.0 with H2SO4) and B [2 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g MgSO4 . 7H2O and 0.1 g KCl in 500 ml distilled water; pH adjusted to 2.7 with H2SO4], which had previously been autoclaved (final pH was approximately 4).

PCR amplification and sequencing of the 16S rRNA gene of the novel strain and phylogenetic analysis were performed as described by Tamura & Hatano (2001)Go. Distances (distance options according to the Kimura two-parameter model) were calculated and clustering was performed by using the neighbour-joining and maximum-parsimony methods; bootstrap values based on 1000 replications were used for this purpose. 16S rRNA gene sequence analysis revealed that strain Aac-30T formed a monophyletic cluster with C. acidiphila (Fig. 1Go). The level of similarity between strain Aac-30T and the type strain of C. acidiphila was 99.4 %.


Figure 1
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  		Fig. 1. Phylogenetic tree derived from 16S rRNA gene sequences of strain Aac-30T and members of the suborder Catenulisporineae. The tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987Go). The 16S rRNA gene sequence of Streptomyces ambofaciens ATCC 23877T was used as the outgroup. Numbers at branch points are confidence limits (expressed as percentages) estimated from a bootstrap analysis of 1000 replicates. Bar, 0.01 Knuc.

 
Analyses of whole-cell sugar patterns, cell-wall amino acids, menaquinones, the acyl type of the peptidoglycan, mycolic acid and DNA base composition were performed as described by Tamura et al. (1994)Go. Processing and analysis of fatty acids as methyl esters were performed by using the protocol of the MIDI Sherlock Microbial Identification System (Sasser, 1990Go). Freeze-dried cells for chemotaxonomic analyses were obtained from a culture grown in yeast extract–glucose broth (1.0 % yeast extract, 1.0 % D-glucose; pH adjusted to 5.5 with H2SO4) on a rotary shaker at 28 °C. The predominant menaquinones of strain Aac-30T were MK-9(H6) and MK-9(H8); MK-9(H4) was present as a minor component. The strain contained LL-A2pm, glycine, alanine and glutamic acid as cell-wall amino acids, and ribose, mannose, arabinose and glucose as whole-cell sugars. The fatty acid profile of strain Aac-30T comprised iso-C16 : 0 (47.9 %), anteiso-C17 : 0 (22.2 %), 9 methyl C16 : 0 (4.7 %), iso-C17 : 0 (4.7 %), anteiso-C17 : 1 (4.4 %), iso-C15 : 0 (3.8 %), anteiso-C15 : 0 (3.2 %), C16 : 0 (3.1 %), iso-C16 : 1 (2.0 %), C16 : 1{omega}9c (1.9 %), C17 : 0 cyclo (1.5 %) and 10 methyl C17 : 0 (1.2 %). Significant differences in the fatty acid profiles of strains Aac-30T and C. acidiphila NBRC 102108T were found only for the proportions of iso-C16 : 0 and anteiso-C17 : 0 (Supplementary Table S1 available in IJSEM Online). As diagnostic phospholipids, phosphatidylglycerol and phosphatidylinositol were detected, but phosphatidylcholine, phosphatidylethanolamine and some other phospholipids containing glucosamine were not detected (phospholipid type PI sensu Lechevalier et al., 1977Go). The DNA G+C content of strain Aac-30T was 69.1 mol%.

Morphological characteristics were determined with the use of scanning electron microscopy as described by Tamura et al. (1994)Go. Strain Aac-30T formed a rectiflexible or straight spore chain consisting of rod-shaped spores with a diameter of 0.7–1.5 µm. Determination of cultural and physiological characteristics was performed as described by Gordon et al. (1974)Go, Seino et al. (1985)Go, Shirling & Gottlieb (1966)Go and Yokota et al. (1993)Go, and by using the API ZYM system (bioMérieux). Detailed results of these analyses are provided in the species description below and in Tables 1Go and 2Go. Strain Aac-30T formed red colonies that developed on most of the acidic media tested. The colour of the aerial mycelia and spore chains ranged from white to pink on International Streptomyces Project (ISP) media 2, 4, 5 and 7 in which the pH was adjusted to approximately 5. This strain grew on media in which the pH was adjusted to 4–6.5 but did not grow on medium with pH adjusted to 7. On acidified ISP medium 9 (Shirling & Gottlieb, 1966Go), good growth was observed in the presence of D-glucose, L-arabinose, D-xylose, fructose, sucrose and raffinose; no visible colonies were observed in the presence of inositol or rhamnose, and scant growth was observed in the presence of mannitol. Positive reactions were found for starch hydrolysis, esterase, trypsin and beta-glucuronidase. The cultural characteristics of strain Aac-30T were clearly different from those of C. acidiphila (Table 2Go).


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  		Table 1. Cultural characteristics of strain Aac-30T

The diffusible pigment was not produced in all media tested. Colour codes in parentheses correspond to those given by Rayner (1970)Go.

 

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  		Table 2. Differential characteristics between strain Aac-30T and Catenulispora acidiphila

Taxa: 1, strain Aac-30T (data from this study); 2, C. acidiphila (data from Busti et al., 2006Go and this study). Both strains utilize D-glucose, L-arabinose, D-xylose and D-fructose, but not myo-inositol or L-rhamnose. Both strains are positive for alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, acid phosphatase, phosphohydrolase, {alpha}-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase and {alpha}-mannosidase but negative for lipase, cystine aminopeptidase, {alpha}-galactosidase and {alpha}-fucosidase. +, Positive; –, negative.

 
The microplate hybridization method developed by Ezaki et al. (1988Go, 1989)Go was applied to determine DNA relatedness. The level of DNA–DNA relatedness between strain Aac-30T and C. acidiphila NBRC 102108T was below 26 %. Strain Aac-30T was also distinguishable from C. acidiphila based on several phenotypic properties (Table 2Go). Therefore, on the basis of morphological, physiological and chemotaxonomic data, DNA–DNA hybridization results and phylogenetic distinctiveness, strain Aac-30T is considered to represent a novel species of the genus Catenulispora, for which the name Catenulispora rubra sp. nov. is proposed.

Description of Catenulispora rubra sp. nov.
Catenulispora rubra (ru'bra. L. fem. adj. rubra red).

Gram-positive, non-acid-fast aerobic actinomycete that forms extensively branched substrate hyphae. The vegetative hypha is finely branched and does not fragment. Monopodially or dichotomously branching aerial mycelia develop sparsely. The aerial mycelium contains spore chains arranged in a hook-like or flexuous manner. Spores (0.7–1.5 µm in diameter) are rod-shaped, smooth-surfaced and non-motile. Contains mannose, arabinose and glucose. Colonies are red-pigmented. The optimum temperature range for growth is 20–30 °C, and optimum pH is 5. D-Glucose, raffinose, L-arabinose, D-xylose, sucrose and fructose, but not inositol or rhamnose, are used as sole carbon sources. Positive for alkaline phosphatase, esterase, leucine aminopeptidase, valine aminopeptidase, acid phosphatase, phosphohydrolase, {alpha}-glucosidase, beta-glucuronidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, {alpha}-mannosidase and trypsin, but negative for lipase, cystine aminopeptidase, {alpha}-galactosidase and {alpha}-fucosidase. Hydrolyses starch and aesculin, but not gelatin or urea. The predominant menaquinones are MK-9(H6) and MK-9(H8). The cellular fatty acids consist of iso-branched, anteiso-branched, saturated and unsaturated fatty acids with iso-C16 : 0 and anteiso-C17 : 0 as major components. The cell wall contains LL-A2pm and glycine (peptidoglycan type A3{gamma}' sensu Schleifer & Kandler, 1972Go). The DNA G+C content is 69.1 mol%.

The type strain, Aac-30T (=NBRC 101179T=DSM 44948T), was isolated from forest soil in Oodate, Japan.


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 MAIN TEXT
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Busti, E., Cavaletti, L., Monciardini, P., Schumann, P., Rohde, M., Sosio, M. & Donadio, S. (2006). Catenulispora acidiphila gen. nov., sp. nov., a novel, mycelium-forming actinomycete, and proposal of Catenulisporaceae fam. nov. Int J Syst Evol Microbiol 56, 1741–1746.[Abstract/Free Full Text]

Ezaki, T., Hashimoto, Y., Takeuchi, N., Yamamoto, H., Liu, S.-L., Miura, H., Matsui, K. & Yabuuchi, E. (1988). Simple genetic identification method of viridans group streptococci by colorimetric dot hybridization and quantitative fluorometric hybridization in microdilution wells. J Clin Microbiol 26, 1708–1713.[Abstract/Free Full Text]

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24, 54–63.[Abstract/Free Full Text]

Lechevalier, M. P., De Bièvre, C. & Lechevalier, H. A. (1977). Chemotaxonomy of aerobic actinomycetes: phospholipid composition. Biochem Syst Ecol 5, 249–260.[CrossRef]

Rayner, R. W. (1970). A Mycological Colour Chart. Kew, UK: Commonwealth Mycological Institute and British Mycological Society.

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids, MIDI Technical Note 101. Newark, DE: MIDI Inc.

Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36, 407–477.[Free Full Text]

Seino, A., Arai, M., Enokita, R., Okazaki, T. & Furuichi, A. (1985). Identification Manual of Actinomycetes. Tokyo: The Society for Actinomycetes Japan (in Japanese).

Shirling, E. B. & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[Medline]

Tamura, T. & Hatano, K. (2001). Phylogenetic analysis of the genus Actinoplanes and transfer of Actinoplanes minutisporangius Ruan et al. 1986 and ‘Actinoplanes aurantiacus’ to Cryptosporangium minutisporangium comb. nov. and Cryptosporangium aurantiacum sp. nov. Int J Syst Evol Microbiol 51, 2119–2125.[Abstract]

Tamura, T., Nakagaito, Y., Nishii, T., Hasegawa, T., Stackebrandt, E. & Yokota, A. (1994). A new genus of the order Actinomycetales, Couchioplanes gen. nov., with descriptions of Couchioplanes caeruleus (Horan and Brodsky 1986) comb. nov. and Couchioplanes caeruleus subsp. azureus subsp. nov. Int J Syst Bacteriol 44, 193–203.[Abstract/Free Full Text]

Yokota, A., Tamura, T., Hasegawa, T. & Huang, L. H. (1993). Catenuloplanes japonicus gen. nov., sp. nov., nom. rev., a new genus of the order Actinomycetales. Int J Syst Bacteriol 43, 805–812.[Abstract/Free Full Text]




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