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1 Korea Research Institutes of Biosciences and Biotechnology, 52 Eoeun-dong, Yuseong gu, Daejeon 305-333, Republic of Korea
2 Rural Resources Development Institute, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 411-853, Republic of Korea
Correspondence
Chang-Jin Kim
changjin{at}kribb.re.kr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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These authors contributed equally to this work. 
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A 9500T is AB049939.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) was the first described member of the family Thermoactinomycetaceae (Matsuo et al., 2006). Recently, based on 16S rRNA gene sequence analysis, as well as chemotaxonomic and physiological characterization, the members of the genus Thermoactinomyces have been divided into six genera, Thermoactinomyces sensu stricto (Yoon et al., 2005), Laceyella (Yoon et al., 2005), Thermoflavimicrobium (Yoon et al., 2005), Seinonella (Yoon et al., 2005), Planifilum (Hatayama et al., 2005) and Mechercharimyces (Matsuo et al., 2006). At the time of writing, the reclassification of the family Thermoactinomycetaceae means that there are now the following recognized species: Thermoactinomyces candidus (Kurup et al., 1975; proposed as a later synonym of Thermoactinomyces vulgaris by Yoon et al. 2000); Thermoactinomyces vulgaris (Tsilinsky, 1899); Thermoactinomyces intermedius (Kurup et al., 1980); Thermoactinomyces thalpophilus (Unsworth & Cross, 1980; proposed as a later synonym of Thermoactinomyces sacchari by Yoon et al. 2000); Thermoflavimicrobium dichotomicus (Krasil'nikov & Agre, 1964; Cross & Goodfellow, 1973); Laceyella sacchari (Lacey, 1971; Yoon et al., 2005; basonym Thermoactinomyces sacchari); Laceyella putida (Lacey & Cross, 1989; Yoon et al., 2005; basonym Thermoactinomyces putidus); Seinonella peptonophila (Nonomura & Ohara, 1971; Yoon et al., 2005; basonym Thermoactinomyces peptonophilus); Planifilum fimeticola (Hatayama et al., 2005); Planifilum fulgidum (Hatayama et al., 2005); Mechercharimyces mesophilus (Matsuo et al., 2006) and Mechercharimyces asporophorigenens (Matsuo et al., 2006). These species are aerobic, Gram-positive and thermophilic, with the exception of two mesophilic species, Seinonella peptonophila and Mechercharimyces mesophilus. Here we describe another mesophilic member of the family Thermoactinomycetaceae.
Strain A 9500T was isolated and maintained on Bennett's agar (Atlas, 1993) medium for 2 weeks at 30 °C. Other media used for cultural characteristics were inorganic salt–starch agar, yeast extract/malt extract agar, oatmeal agar, glycerol asparagine agar and tyrosine agar (Shirling & Gottlieb, 1966). The morphological characteristics of 7 day cultures of strain A 9500T grown on Bennett's agar were observed by light microscopy (JEOL) and scanning electron microscopy (SEM 515; Philips). Morphological studies revealed that the new isolate shared the characteristics already described for the family Thermoactinomycetaceae, with white aerial mycelium and pale yellow substrate mycelium which was well developed. Substrate mycelium was branched, but not fragmented. Spores formed singly (1.0–1.4x0.7–0.9 µm) on aerial mycelium and were non-motile (Fig. 1a). Substrate hyphae were sessile and had extensively branched (0.3–0.6 µm) sporophores with the structure and properties of bacterial endospores (Fig. 1b). The colours of the substrate and aerial mycelia and any soluble pigments were determined (Kelly, 1964). Strain A 9500T grew on all media tested. Good growth was observed on Bennett's agar and no diffusible pigments were produced on the media tested.
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. Other physiological features and carbon source utilization were observed on a basal medium used for cultivation of members of the genus Thermoactinomyces (Williams et al., 1989). Detailed physiological and biochemical characteristics of the novel isolate are given in Table 1 and in the species description.
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and Schleifer & Kandler (1972) using cellulose TLC plates. The menaquinone and cellular fatty acid profiles were determined as described by Collins et al. (1977) and were analysed by HPLC (Kroppenstedt, 1982). The cell-wall peptidoglycan of the novel isolate contained meso-diaminopimelic acid (meso-DAP), glutamic acid and alanine, but no diagnostic sugars. Thus strain A 9500T had an unusual type III cell wall (Lechevalier & Lechevalier, 1970). This result was consistent with that obtained previously for other members of the family Thermoactinomycetaceae. The diagnostic phospholipid was phosphatidylethanolamine. However, the predominant menaquinones were MK-9(H4) and MK-10(H4) in a ratio of 7 : 3. For the above analysis, the organism was cultivated on Bennett's broth for 3 days at 32 °C and harvested by centrifugation at 3000 r.p.m. for 20 min. Cellular fatty acid analysis was performed as described by Sasser (1990) using the Microbial Identification System (MIDI). The fatty acid profile of strain A 9500T was mainly composed of anteiso-C15 : 0 (43.34 %), iso-C16 : 0 (14.23 %), C16 : 0 (7.90 %), iso-C15 : 0 (7.40 %), anteiso-C17 : 0 (7.17 %), iso-C14 : 0 (6.14 %), C15 : 0 (4.01 %) and anteiso-C16 : 0 (3.76). The G+C DNA content of strain A 9500T was determined by using the HPLC method of Mesbah et al. (1989) and was 39.4 mol%.
Extraction and amplification of genomic DNA for 16S rRNA gene sequence analysis was carried out as described by Cui et al. (2001). The 16S rRNA gene fragment was amplified by using universal primers corresponding to positions 8–27 for the forward primer and 1492–1510 for the reverse primer (Escherichia coli numbering system; Weisburg et al., 1991). Based on 1459 bp long 16S rRNA gene sequences, phylogenetically related bacteria were aligned by using a BLAST search (Altschul et al., 1990) against the GenBank database. Multiple alignments with sequences of related taxa of the family Thermoactinomycetaceae were implemented by using CLUSTAL_X (Thompson et al., 1997). The 16S rRNA gene sequence similarity values were calculated by pairwise comparison (Kimura, 1980). Phylogenetic trees (Fig. 2) were constructed based on different tree-making algorithms, namely the neighbour-joining (Saitou & Nei, 1987), least squares (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993) and maximum-parsimony (Fitch, 1971) methods. A neighbour-joining tree was reconstructed from evolutionary distances calculated using the Jukes–Cantor coefficient (Jukes & Cantor, 1969). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.
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) with Laceyella putida KCTC 3666T. The highest 16S rRNA gene similarity values found between strain A 9500T and other genera of the family Thermoactinomycetaceae were with Thermoactinomyces vulgaris KCTC 9557 (90.38 % gene sequence similarity), Thermoflavimicrobium dichotomicum KCTC 3667T (90.22 %), Laceyella sacchari KCTC 9790T (90.04 %), Thermoactinomyces intermedius KCTC 9646T (89.98 %), Laceyella putida KCTC 3666T (89.67 %), Mechercharimyces asporophorigenens DSM 44955T (89.50 %), Mechercharimyces mesophilus DSM 44894T (89.41 %), Seinonella peptonophila KCTC 9740T (89.41 %), Planifilum fimeticola JCM 12507T (88.43 %) and Planifilum fulgidum JCM 12508T (88.35 %). The length of sequence compared was 1459 bp.
It is evident from the gene sequence similarity values, the phylogenetic tree (Fig. 2) and the phenotypic properties (Table 1) that strain A 9500T can be clearly distinguished from other genera of the family Thermoactinomycetaceae. Examination of the growth temperature, DNA G+C contents, menaquinone composition, cellular fatty acid content and the ability to degrade gelatin reveals that strain A 9500T does not belong to the genera Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium, Seinonella, Planifilum or Mechercharimyces. On the basis of the results presented in this study, strain A 9500T has been assigned to a novel genus in the family Thermoactinomycetaceae, for which the name Shimazuella kribbensis gen. nov., sp. nov. is proposed.
Description of Shimazuella gen. nov.
Shimazuella (Shi.ma.zu.el'la. N.L. fem. n. Shimazuella named after Akira Shimazu, a Japanese microbiologist from Tokyo University, who has contributed to the field of prokaryotic taxonomy).
Cells are Gram-positive, aerobic and mesophilic. Aerial mycelium is abundant and white and is not fragmented. Forms well-developed, branched and septate substrate mycelia on Bennett's agar and yeast extract-malt extract (ISP 2). Soluble pigments are not produced. The cell-wall peptidoglycan contains meso-diaminopimelic acid, glutamic acid and alanine, but no characteristic sugars. Endospores are produced singly along mycelia. The major menaquinone is MK-9 and is found at a ratio of 7 : 3 with MK-10. Major fatty acids are anteiso-C15 : 0, iso-C16 : 0, C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The G+C content is 39.4 mol%. The type species is Shimazuella kribbensis.
Description of Shimazuella kribbensis sp. nov.
Shimazuella kribbensis (krib.ben'sis. N.L. fem. adj. kribbensis pertaining to KRIBB, an arbitrary adjective formed from the acronym of the Korea Research Institute of Bioscience and Biotechnology, KRIBB, where the taxonomic studies on this new genus and novel species were performed).
In addition to the genus description, exhibits the following characteristics. Colonies are fast-growing, ridged with white mycelia and a feathery margin on Bennett's agar at 28 °C. Growth occurs between 20 and 37 °C, with optimum growth at 32 °C. Forms endospores singly on unbranched sporophores. Casein and starch are degraded, but not gelatin, hypoxanthine, xanthine or L-tyrosine. No pigments are observed on all media tested. Growth occurs in the presence of 25 µg novobiocin ml–1. Major cellular fatty acids are anteiso-C15 : 0, iso-C16 : 0, C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The DNA G+C content of the type strain is 39.4 mol%.
The type strain, A 9500T (=KCTC 9933T=DSM 45090T), was isolated from a soil sample collected from Sobaek mountain, Republic of South Korea.
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ACKNOWLEDGEMENTS |
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