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1 Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
2 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
3 Institute of Hyperthermophiles, Motobu-Noge Hospital, Aza-Ohama 880-1, Motobu, Okinawa 905-0212, Japan
Correspondence
Akira Nakamura
a-nak{at}agbi.tsukuba.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Planifilum fimeticola H0165T and Planifilum fulgidum 500275T, C0170 and 700375 are AB088364, AB088362, AB088363 and AB088361, respectively.
Tables detailing levels of DNA–DNA relatedness among the novel strains and cellular fatty acid profiles of the four strains plus representative members of the family ‘Thermoactinomycetaceae’ are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). However, based on 16S rRNA gene sequence analysis, as well as chemotaxonomic and physiological characterization of the genus, Yoon & Park (2000) suggested that Thermoactinomyces was a heterogeneous group containing more than one genus. Based on further chemotaxonomic characterization, it was proposed that this genus should be divided into four genera, Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium and Seinonella (Yoon et al., 2005). The members of this family are aerobic, Gram-positive, thermophilic bacteria, except the mesophilic genus Seinonella, and show filamentous growth like actinomycetes. Members of the four genera have been isolated from a variety of natural sources, such as mushroom compost (Song et al., 2001), moulding hay, cereal grains, soil, river water, dairy products and marine sediments, and also from humidifiers of air-conditioning systems (Lacey & Cross, 1989).
In our previous study to characterize the microbial flora in a hyperthermal composting process in Japan (Hatayama et al., 2005), we isolated four thermophilic bacteria showing filamentous growth at approximately 65 °C. From phylogenetic analyses based on 16S rRNA gene sequences as well as chemotaxonomic, physiological and phenotypic characterizations of these bacteria, we propose that they constitute a new genus in the family ‘Thermoactinomycetaceae’, consisting of two species.
Strains H0165T, 500275T, C0170 and 700375 were isolated from samples of the hyperthermal composting process (Hatayama et al., 2005) by cultivation on Luria–Bertani (LB) agar plates at 65 °C. Growth of strain H0165T was observed at 50–65 °C with an optimum at 55–63 °C, and the other three strains grew at 50–67 °C with optima at 60–65 °C in liquid LB medium. Morphological characteristics of these strains resembled those of the genus Thermoactinomyces sensu stricto in formation of substrate mycelia with single spores along the mycelia (Fig. 1). However, formation of aerial mycelia was not observed in any of the isolates grown on LB agar, CYC agar (Lacey & Cross, 1989), SY agar (Yoon et al., 2005) or Bacto nutrient agar (Difco); colonies were lustrous, cream-yellow with radial wrinkles. This is at variance with the description that species in the family ‘Thermoactinomycetaceae’ form aerial mycelia on plates of at least one of these agars (Lacey & Cross, 1989).
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). From searches using BLASTN (Altschul et al., 1997) and FASTA (Pearson, 2000), the four isolates showed closest 16S rRNA gene sequence similarities to the unidentified low-G+C, Gram-positive bacterium HTA1417 (94·2–94·8 % similarity), which was isolated from the deepest sea mud of the Mariana Trench (Takami et al., 1997). The closest recognized species to the isolates were those of the genus Thermoactinomyces sensu stricto (91·2–91·6 % 16S rRNA gene sequence similarity), Laceyella (88·8–89·4 %), Thermoflavimicrobium (89·3–89·7 %) and Seinonella (87·8–88·1 %). To visualize the phylogenetic relationship between the new isolates and these four genera, two phylogenetic trees were constructed using the maximum-likelihood (data not shown) and neighbour-joining (Fig. 2) methods. The two methods produced essentially the same phylogenetic trees, and indicated that the four isolates formed an independent cluster close to that of the genera Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium and Seinonella with a high bootstrap value (Fig. 2). This indicated that the isolates should be classified within a novel genus of the family ‘Thermoactinomycetaceae’. Moreover, the analyses suggested that the isolates could be divided into two genomic groups, H0165T and the other three strains (97·1–97·6 % sequence similarity between the two groups). This split was confirmed by the results of DNA–DNA hybridization experiments, determined with the method of Ezaki et al. (1989) (see Supplementary Table S1 available in IJSEM Online).
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. The values obtained were over 10 mol% higher than those of the other genera of the family ‘Thermoactinomycetaceae’ (Table 1).
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using cellulose HPTLC plates (Merck). Profiles of menaquinones and cellular fatty acids, conducted by the National Collections of Industrial and Marine Bacteria (NCIMB), Japan, Co. Ltd, were determined by using the methods of Tamaoka (1986) and Nishijima et al. (1997), and Yoon et al. (2005), respectively. The cell-wall peptidoglycan of the isolates contained meso-diaminopimelic acid, alanine and glutamic acid but no diagnostic sugars, indicating that the wall chemotype was of type III (Lechevalier & Lechevalier, 1970). This result was consistent with those of the members of the family ‘Thermoactinomycetaceae’. However, the isolates contained almost only one menaquinone type, MK-7 (>98·1 % peak area ratio), which distinguished them from the genera Thermoactinomyces sensu stricto, Laceyella and Seinonella (Table 1
). Moreover, the cellular fatty acid profiles of the isolates differed from those of the other four genera (Table 1 and Supplementary Table S2 in IJSEM Online). In particular, the new isolates contained longer branched cellular fatty acids, such as iso-C17 : 0 and anteiso-C17 : 0, as major components compared with the type species of the four genera. In addition, differences in the major components of the cellular fatty acids were seen between strains H0165T and 500275T.
Utilization of sugars was tested using the methods of Shirling & Gottlieb (1966) and Fortina et al. (2001). No reproducible results were obtained because of the weak growth of the strains (data not shown). Degradation of substrates was tested using LB agar with 1 % casein, 1 % starch, 1 % avicel, 1 % carboxymethyl cellulose, 1 % xylan, 0·5 % hypoxanthine or 0·5 % xanthine, and using CYC agar with 0·5 % L-tyrosine. All the isolates degraded casein and starch but not other substrates, except that strain H0165T was able to degrade L-tyrosine (Table 1).
Strains H0165T, 500275T, C0170 and 700375 were clearly distinguished from members of the related genera Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium and Seinonella based on the absence of aerial mycelia, growth temperatures, DNA G+C contents and compositions of menaquinones and cellular fatty acids. Phylogenetic analyses based on the 16S rRNA gene sequences showed that the isolates constituted an independent cluster close to that of these four genera. The results indicated that the isolates did not belong to Thermoactinomyces sensu stricto, Laceyella, Thermoflavimicrobium or Seinonella. Moreover, strain H0165T could be clearly discriminated from the other three isolates based on 16S rRNA gene sequence similarities, levels of DNA–DNA relatedness, the cellular fatty acid profile and the ability to degrade L-tyrosine. On the basis of these results, the four isolates described herein have been assigned to two species of a novel genus in the family ‘Thermoactinomycetaceae’, for which the names Planifilum fimeticola gen. nov., sp. nov. (H0165T) and Planifilum fulgidum sp. nov. (500275T, C0170 and 700375) are proposed.
Description of Planifilum gen. nov.
Planifilum (Pla.ni.fi'lum. L. adj. planus flat; L. neut. n. filum a thread; N.L. neut. n. Planifilum a flat thread).
Cells are Gram-positive, aerobic and thermophilic. Substrate mycelia are formed on LB agar, CYC agar, SY agar and Bacto nutrient agar, but aerial mycelia are not formed. Endospores are produced singly along mycelia. The cell-wall peptidoglycan contains meso-diaminopimelic acid, alanine and glutamic acid but no diagnostic sugars. The major menaquinone is MK-7. Major fatty acids are iso-C17 : 0, anteiso-C17 : 0 and iso-C15 : 0 or C16 : 0. DNA G+C content is 58·7–60·3 mol%. The type species is Planifilum fimeticola.
Description of Planifilum fimeticola sp. nov.
Planifilum fimeticola (fi.me.ti.co'la. L. n. fimetum a dung-hill and, by extension, compost; L. masc. suffix -cola inhabitant; N.L. masc. n. fimeticola inhabitant of compost, referring to the habitat of the type strain).
Colonies are lustrous, cream-yellow with radial wrinkles. Growth occurs at 50–65 °C, optimally at 55–63 °C. Casein, starch and L-tyrosine are degraded. The major cellular fatty acids are iso-C16 : 0, iso-C17 : 0 and anteiso-C17 : 0. DNA G+C content of the type strain is 60·3 mol%.
The type strain, H0165T (=ATCC BAA-969T=JCM 12507T), was isolated from a hyperthermal composting process in Okinawa prefecture, Japan.
Description of Planifilum fulgidum sp. nov.
Planifilum fulgidum (ful'gi.dum. L. neut. adj. fulgidum lustrous, referring to the colony character).
Colonies are lustrous, cream-yellow with radial wrinkles. Growth occurs at 50–67 °C, optimally at 60–65 °C. Casein and starch are degraded. The major cellular fatty acids are iso-C17 : 0, anteiso-C17 : 0 and iso-C15 : 0. The DNA G+C content is 58·7–60·0 mol%.
The type strain, 500275T (=ATCC BAA-970T=JCM 12508T), was isolated from a hyperthermal composting process in Okinawa prefecture, Japan. Two additional strains, C0170 and 700375, are included in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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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.
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