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Int J Syst Evol Microbiol 57 (2007), 1851-1854; DOI  10.1099/ijs.0.64646-0
© 2007 International Union of Microbiological Societies

Planifilum yunnanense sp. nov., a thermophilic thermoactinomycete isolated from a hot spring

Yan-xin Zhang, Chen Dong and Shen Biao

Key Laboratory of Microbiological Engineering of Agricultural Environment, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China

Correspondence
Biao Shen
shenbiao{at}njau.edu.cn


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A thermophilic actinomycete, strain LA5T, was isolated from a hot spring in Yunnan province, People's Republic of China. The isolate grew aerobically at temperatures of 50–75 °C. Aerobic mycelia were not observed. Single spores were produced along the substrate hypha. Strain LA5T was characterized chemotaxonomically by having MK-7 as the predominant menaquinone and iso-C17 : 0 (27.7 %) and C16 : 0 (22.4 %) as the major fatty acids. The cell wall contained meso-diaminopimelic acid. The DNA G+C content was 56.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain LA5T belongs to the genus Planifilum. Levels of DNA–DNA relatedness between strain LA5T and Planifilum fimeticola H0165T and Planifilum fulgidum 500275T were 43.6 and 52.7 %, respectively. On the basis of phenotypic properties and phylogeny, strain LA5T represents a novel species of the genus Planifilum, for which we propose the name Planifilum yunnanense sp. nov., with the type strain LA5T (=CCTCC AA206002T=KCTC 13052T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LA5T is DQ119659.


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Only one genus, Thermoactinomyces, was included in the family ‘Thermoactinomycetaceae’ before 2005. Yoon et al. (2005)Go suggested that the family ‘Thermoactinomycetaceae’ should be divided into four genera, Laceyella, Thermoactinomyces sensu stricto, Thermoflavimicrobium and Seinonella, based on 16S rRNA gene sequence analysis as well as chemotaxonomic and physiological characteristics of recognized Thermoactinomyces species. In addition, Hatayama et al. (2005)Go proposed a new genus Planifilum with two species.

Strain LA5T was isolated from a hot spring in Yunnan province, People's Republic of China. It could grow at temperatures of 50–75 °C, the highest growth temperature obtained for all Thermoactinomyces strains reported so far, with optimum growth occurring at 60–70 °C. Colonies of 3–4 mm in diameter appeared after 48 h incubation on LB Gelrite plates at 70 °C. The colonies were cream–yellow with radial wrinkles. No aerobic mycelia were observed. Single spores were produced along the substrate hypha. Strain LA5T did not produce any diffusible pigment during the whole incubation period. Strain LA5T grew at pH 6–10, with optimum growth at pH 8.5.

To establish accurately the phylogenetic position of strain LA5T, its 16S rRNA gene sequence was determined. Genomic DNA extraction and PCR amplification of 16S rRNA genes using universal primers were performed by using the method of Ausubel et al. (1999)Go. The resultant sequence for strain LA5T was aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred using minimum-evolution, maximum-parsimony and neighbour-joining methods (Kumar et al., 2004Go). The bootstrap values of the neighbour-joining tree were calculated based on 1000 repeats.

The results of alignment with 16S rRNA gene sequences obtained from GenBank showed that strain LA5T had similarities of 99 and 97 % with Planifilum fulgidum 500275T and Planifilum fimeticola H0165T, respectively, and less than 94 % with any others used. In the phylogenetic trees constructed using three different methods, strain LA5T and members of the genus Planifilum formed an independent cluster in the family ‘Thermoactinomycetaceae’. Strain LA5T was most closely related to P. fulgidum 500275T, as shown in the neighbour-joining tree (Fig. 1Go). Therefore, strain LA5T should be classified in the genus Planifilum.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of strain LA5T, members of the family ‘Thermoactinomycetaceae’ and Bacillus subtilis DSM 618. Numbers at nodes are percentage bootstrap values based on 1000 repeats (only values greater than 50 % are shown). Bar, 0.01 substitutions per nucleotide position.

 
The DNA base composition of strain LA5T was determined by using the thermal denaturation method (Marmur & Doty, 1962Go), with Escherichia coli K-12 as reference (DNA G+C content, 50 mol%). The DNA G+C content was calculated by using the method of De Ley (1970)Go. The results showed that the DNA G+C content of strain LA5T was 56.8 mol%, close to the values obtained for P. fulgidum 500275T (60.0 mol%) and P. fimeticola H0165T (60.3 mol%), but much higher than those of other members of the family ‘Thermoactinomycetaceae’, which also indicated that strain LA5T was closely related to the genus Planifilum.

In order to determine further the relationship between strain LA5T and other members of the genus Planifilum phylogenetically, DNA–DNA hybridizations of strain LA5T with P. fulgidum 500275T and P. fimeticola H0165T were respectively performed by using thermal denaturation and reassociation (De Ley et al., 1970Go). A UV spectrophotometer (Lambda 35; Perkin Elmer) and temperature controller (PTP-6; Perkin Elmer) were used. Levels of DNA–DNA relatedness between strain LA5T and P. fimeticola H0165T and P. fulgidum 500275T were only 43.6 and 52.7 %, respectively. Therefore, strain LA5T represents a novel species of the genus Planifilum.

The chemotaxonomic characteristics of strain LA5T were determined using cells cultured in LB medium, supplemented with starch. Cell walls were analysed for principal amino acids by using the method of Lechevalier & Lechevalier (1970)Go. Whole-cell sugars were determined using GC and MS (GC-MS model HP-6890; Hewlett Packard) according to the methods of Saddler et al. (1991)Go and Takahashi & Egusa (1992)Go. Cellular fatty acids were analysed using HP-6890 GC, equipped with a hydrogen flame-ionization detector (FID) and HP Chemstation (version A 5.01), based on the method of Collins (1985)Go. The results revealed the presence of meso-diaminopimelic acid, alanine and glutamic acid, indicating that the cell wall chemotype was type III, consistent with all other members of the family ‘Thermoactinomycetaceae’. MK-7 was the predominant menaquinone. Whole-cell sugars were found to be glucose and ribose, without any diagnostic sugars. The predominant fatty acids were iso-C17 : 0 (27.7 %), C16 : 0 (22.4 %), iso-C16 : 0 (8.8 %) and anteiso-C17 : 0 (8.6 %), which were different from those of the type strains of species of all five genera in the family ‘Thermoactinomycetaceae’ (Table 1Go).


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  		Table 1. Phenotypic characteristics of strain LA5T and recognized Planifilum species

Strains: 1, P. fimeticola H0165T; 2, P. fulgidum 500257T; 3, strain LA5T (Planifilum yunnanense sp. nov.). +, Positive reaction; –, negative reaction; NO, not observed. All strains can use the following substrates as carbon sources: casein, glutin and starch, {alpha}-cyclodextrin, D-fructose, maltose, D-mannitol, D-raffinose, D-melibiose, turanose, D-gluconic acid, beta-hydroxybutyric acid, {gamma}-hydroxybutyric acid, {alpha}-ketovaleric acid, malonic acid, propionic acid, acetic acid, cis-aconitic acid, D-alanine and L-alanine.

 
Utilization of sugars and other substrates was tested using the following medium (g l–1): (NH4)2SO4, 1.5; KH2PO4, 0.5; K2HPO4 . 3H2O, 1.5; MgSO4 . 7H2O, 0.2; NaCl, 0.5; yeast extract, 0.02; at 65 °C and pH 8.5. Strain LA5T could utilize many sugars, organic acids and amino acids as sole carbon sources and showed a greater range of substrate utilization than P. fimeticola H0165T and P. fulgidum 500257T (Table 1Go). Strain LA5T grew on LB, potato dextrose agar, Czapek and starch-casein, but only weak growth was observed on glycerol-asparagine and Gause's synthetic medium.

Description of Planifilum yunnanense sp. nov.
Planifilum yunnanense (yun.nan.en'se. N.L. neut. adj. Yunnanense referring to Yunnan, the southern Province, P. R. China, where the sample was collected).

Colonies are rough and cream–yellow with radial wrinkles. Growth occurs at 50–75 °C (optimum growth at 60–70 °C) and pH 6.0–10.0 (optimum growth at pH 8.5). Casein, glutin and starch are degraded. Predominant fatty acids are iso-C17 : 0 (27.7 %) and C16 : 0 (22.4 %). The DNA G+C content of the type strain is 56.8 mol%.

The type strain, LA5T (=CCTCC AA206002T=KCTC 13052T), was isolated from a hot spring in Yunnan Province, P. R. China.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Professor Akira Nakamura (University of Tsukuba, Japan) for kindly providing P. fulgidum 500275T and P. fimeticola H0165T. We also thank Dr Shu-Kun Tang (Yunnan University, China) for analysis of cell-wall components and Dr Xinhua Chen (China Agricultural University, China) for DNA–DNA hybridization.


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Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1999). Short Protocols in Molecular Biology, 4th edn. John Wiley & Sons.

Collins, M. D. (1985). Isoprenoid quinone analysis in bacterial classification and identification. In Chemical Methods in Bacterial Systematics, pp. 267–287. London: Academic Press.

De Ley, J. (1970). Reexamination of the association between melting point, buoyant density, and chemical base composition of DNA. J Bacteriol 101, 738–754.[Abstract/Free Full Text]

De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]

Hatayama, K., Shoun, H., Ueda, Y. & Nakamura, A. (2005). Planifilum fimeticola gen. nov., sp. nov. and Planifilum fulgidum sp. nov., novel members of the family ‘Thermoactinomycetaceae’ isolated from compost. Int J Syst Evol Microbiol 55, 2101–2104.[Abstract/Free Full Text]

Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.[Abstract/Free Full Text]

Lechevalier, M. P. & Lechevalier, H. (1970). Chemical composition as a criterion in the classification of aerobic actinomycetes. Int J Syst Bacteriol 20, 435–443.[Abstract/Free Full Text]

Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, 109–118.[Medline]

Saddler, G. S., Tavecchia, P., Lociure, S., Zanol, M., Colombo, L. & Selva, E. (1991). Analysis of madurose and other actinomycete whole cell sugars by gas chromatography. J Microbiol Methods 14, 185–191.[CrossRef]

Takahashi, Y. & Egusa, H. (1992). Quantitative analysis for madurose and other sugars in small amount of actinomycete whole cells by gas-liquid chromatography. Actinomycetologica 6, 69–78.

Yoon, J.-H., Kim, I.-G., Shin, Y.-K. & Park, Y.-H. (2005). Proposal of the genus Thermoactinomyces sensu stricto and three new genera, Laceyella, Thermoflavimicrobium and Seinonella, on the basis of phenotypic, phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 55, 395–400.[Abstract/Free Full Text]





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