Development of a Simple-Identification Method For

Actinomycetologica (2008) 22:30–33 Copyright Ó 2008 The Society for Actinomycetes Japan VOL. 22, NO. 1 Award Lecture Development of a simple-identiﬁcation method for actinomycetes based on partial 16S rDNA sequences as exempliﬁed by a comparative study of Malaysian and Japanese actinomycetes

Hideyuki Muramatsu Fermentation Research Labs., Astellas Pharma Inc., 5-2-3, Tokodai, Tsukuba, Ibaraki 300-2698, Japan (Received May 14, 2008 / Accepted May 14, 2008 / Published Jun. 25, 2008)

Introduction reasons for this is the nature of the bacterial identification method described above. The use of molecular phyloge- Traditionally, identification of bacteria, including actino- netic data is the most promising solution to this problem mycetes, has been very time consuming and laborious, and, in particular the simplification and cost-minimisation except for some pathogenic species for which simple of procedures are aimed for. identification methods have been established. With most animals and plants, real-time identification of many Development of a simple-identification method for individuals is possible with the naked eye or optical- actinomycetes based on partial 16S rDNA sequences microscopic observation. For bacterial identification, in- cubation prior to morphological observation and various We developed a simple-identification method for actino- tests for phenotypic characteristics are needed. These mycetes based on direct polymerase chain reaction (PCR) difficulties have affected both basic and applied research amplification, direct sequencing, and BLAST homology on bacteria. Bacteria have few morphological character- search (Muramatsu et al., 2003; DNA Data Bank of Japan; istics that can be used to estimate phylogenetic relation- Altschul et al., 1997). It may be said that this method is ships. Molecular phylogenetic methods were applied to in concept similar to DNA barcoding. In previous reports bacterial taxonomy earlier than for other organisms (Olsen of direct PCR amplification of Streptomyces DNA, intact et al., 1994; Larsen et al., 1993). Nucleotide sequences for mycelia grown on the agar media were used as the DNA the 16S ribosomal RNA (rRNA) gene have been published template source (Ishikawa et al., 2000; Tsuchizaki et al., for almost all actinomycete species, and reference to these 2001). We employed liquid cultures in 96-well format sequences allows for easy identification of a wild strain. plates as templates. This enabled the use of multi-pipettes, However, DNA sequencing and analysis involves many which permitted simultaneous treatment of many strains. steps, requires expensive equipment and chemical reagents, The success of the method mainly depended on the success and is particularly difficult when many strains are to of PCR amplification, which in turn was indicated to be analyzed. Fortunately, advances in DNA sequencing depend strongly on the template broth conditions. The technology and high-through-put analytic methods are identification success rate (number of strains identified/ achieved every year. Furthermore, the potential for ‘DNA total number of strains analyzed) among Malaysian and barcoding’ of animals and plants has gained in popularity Japanese isolates were 70.0% and 87.0%, respectively, in recent years (Ratnasingham & Hebert, 2007). Thus, when the ISP-1 based glycine-added medium was used. It methods based on DNA sequencing have the potential to be was observed that Malaysian isolates tended to produce the most generally applicable for identification of not only pigments in the culture broth that may inhibit the bacteria but also plants, animals, and other taxa in the near PCR. Among Vietnamese actinomycete strains, the iden- future. tification rate was 84% (421/500) with the same medium. In natural product-based drug discovery programs, the The rate rose to 96% (480/500) with concentrated nutrient chemical diversity and novelty of the screening sources are broth in phosphate buffer (unpublished data). Nested PCR important. Expanding the taxonomic diversity of isolates contributed to improvement of both the sensitivity and might lead to expansion of the chemical diversity. The specificity. The lower concentration of the primers in the utility of tropical actinomycetes is expected, but presently second PCR mixture enabled performance of the sequenc- there are too few data (Wang et al., 1999). With regard to ing reaction without any purification of the PCR products. basic research on actinomycetes, few distributional studies, About 600 bases of 16S rRNA gene sequence were which should form the foundation of ecological investiga- determined (nucleotide positions 48–653 based on the tions, have been undertaken. One of the most important sequence for Streptomyces ambofaciens, Pernodet et al.,

Corresponding author; Phone: (+81)-29-847-8611. Fax: (+81)-29-847-8313. E-mail: [email protected]

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Table 1. Actinomycetes found in Malaysia and Japan Number of isolates Number of species family genus Japan Malaysia Japan Malaysia Both (Japan Only) (Malaysia Only) Corynebacteriaceae Corynebacterium 0 1 0(0) 1(1) 0 Mycobacteriaceae Mycobacterium 0 4 0(0) 2(2) 0 Nocardiaceae Nocardia 24 23 17(12) 10(5) 5 Rhodococcus 1 2 1(1) 2(2) 0 Geodermatophilaceae Blastococcus 1 0 1(1) 0(0) 0 Micromonosporaceae Micromonospora 10 17 8(8) 11(11) 0 Actinoplanes 21 13 15(14) 9(8) 1 Catellatospora 0 3 0(0) 2(2) 0 Couchioplanes 0 1 0(0) 1(1) 0 Dactylosporangium 1 15 1(1) 10(10) 0 Pilimelia 0 1 0(0) 1(1) 0 Verrucosispora 0 13 0(0) 2(2) 0 Virgosporangium 1 0 1(1) 0(0) 0 Nocardioidaceae Hongia 20 3 6(4) 2(0) 2 Pseudonocardiaceae Amycolatopsis 33 5 11(10) 3(2) 1 Kibdelosporangium 0 1 0(0) 1(1) 0 Actinosynnemataceae Saccharothrix 1 0 1(1) 0(0) 0 Streptomycetaceae Streptomyces 672 541 101(71) 90(60) 30 Kitasatospora 10 1 5(5) 1(1) 0 Streptosporangiaceae Streptosporangium 136 20 18(16) 9(7) 2 Acrocarpospora 3 0 2(2) 0(0) 0 Herbidospora 2 0 2(2) 0(0) 0 Microbispora 1 14 1(1) 3(3) 0 Microtetraspora 1 22 1(0) 2(1) 1 Nonomuraea 4 69 3(0) 14(11) 3 Planotetraspora 31 8 5(3) 3(1) 2 Thermomonosporaceae Actinomadura 6 11 5(4) 5(4) 1 ? Cryptosporangium 1 0 1(1) 0(0) 0 ? Kutzneria 1 0 1(1) 0(0) 0 ? undescribed genus? 0 2 0(0) 1(1) 0 Total 981 790 207(159) 185(137) 48

1989). A sequence homology value of 94% was employed A comparative study of Malaysian as a genus determination criterion, because the intrageneric and Japanese actinomycetes homology exceeds 94% in most genera (Goodfellow et al., 1997; Koch et al., 1996; Lee et al., 2000; Wang et al., About 1,000 strains each of Malaysian and Japanese 1996a; Wang et al., 1996b; Yassin et al., 1997; Zhang et actinomycete were compared using the method (Mura- al., 1997; Zhang et al., 1998; Zhou et al., 1998). In some matsu et al., 2003). Malaysian isolates belonged to 9 cases, we confirmed the genera of isolates from their families, 23 genera, and 185 species, including one genus positions on the phylogenic trees. The number of the as yet undescribed. The Japanese isolates belonged to 9 species was estimated based on a homology value 98.6% as families, 22 genera, and 207 species (Table 1). The fre- criterion consistent with the study of Keswani & Whitman quencies of the families in both geographic areas were (2001). similar, but the frequencies of the genera in each area Simple-identification of thousands of strains is possible differed greatly. There was little taxonomic overlap be- using this method. In this study, the method was used to tween the Malaysian isolates and Japanese isolates. Only evaluate the usefulness of Malaysian actinomycetes as 14% of the species and 50% of the genera occurred in both screening sources of natural product-based drug discovery groups. These results indicate that actinomycetes of greater programs, and it is hoped that it will be greatly useful in taxonomic diversity can be obtained when gathered from distributional studies and the search for new taxa, for both regions, rather than from one or the other. Among the example. Malaysian isolates, two strains were found that apparently

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Actinosynnema mirum IFO14064 T (D85475) 0.01 Knuc Nocardia asteroides ATCC 19247 T (Z36934) T 61.3 90.6 Gordonia bronchialis CIP 1780.88 (X81919) 83.6 Corynebacterium diphtheriae NCTC 11397 T (X84248) Pseudonocardia thermophila IMSNU 20112 T (AJ252830) Micromonospora chalcea DSM 43026 T (X92594) Microsphaera multipartita JCM 9543 T (Y08541) Geodermatophilus obscurus DSM 43161 T (X92355) Sporichthya polymorpha IFO 12702 T (AB025317) 100.0 413C05 (AB123060) 79.4 413C04 (AB123059) Thermobifida alba ATCC 27644 T (AF028247) T 96.6 Streptomonospora salina YIM 90002 (AF178988) 69.4 Nocardiopsis dassonvillei DSM 43111 T (X97886) Thermomonospora chromogena ATCC 43196 T (AF028246) Nonomuraea pusilla IFO 14684 T (U48978) Streptosporangium roseum DSM 43021 T (X89947) T 65.5 72.6 Planobispora longispora IFO 13918 (D85494) 60.4 58.6 85.5 Planomonospora parontospora subsp. parontospora IFO 13880 T (D85495) Microbispora rosea subsp. rosea IFO 14044 T (D86936) T 83.6 Acrocarpospora pleiomorpha IFO 16266 (AB025318) Planotetraspora mira IFO 15435 T (D85496) 50.4 Herbidospora cretacea IFO15474 T (D85485) Actinomadura spadix JCM 3146 T (AF163120) Actinomadura madurae JCM 7436 T (U58527) Thermomonospora curvata JCM 3096 T (AF002262) Streptomyces ambofaciens ATCC 23877 T (M27245) 52.0 Nocardioides jensenii KCTC 9134 T (AF005006) Microlunatus phosphovorus DSM 10555 T (Z78207) 95.4 Luteococcus japonicus IFO12422 T (D85487) Propionibacterium freudenreichii subsp. shermanii DSM 4902 T (Y10819) Bifidobacterium bifidum ATCC 29521 T (M38018)

Fig. 1. Phylogenetic tree showing the position of strains 413C04 & 413C05 based on partial sequence of 16S rDNA (ca. 560 nucleotides, nucleotide positions 89–653 based on the Streptomyces ambofaciens sequence) The numbers below nodes are bootstrap percentages estimated from a bootstrap analysis with 1000 replicates (only values exceeding 50% are indicated). belong to an undescribed genus. Phylogenetic analysis undertake this research. I thank Ms. Mayumi Sodeyama, indicated that they belong to the suborder Streptosporan- Ms. Yosie Aishima, and Ms. Keiko Takeda for technical gineae (Figure 1). The existence of candidate strains of a assistance. novel genus suggests that Malaysian actinomycetes have not been thoroughly investigated and therefore have REFERENCES potential as a source of novel bioactive compounds. These results not only suggested the usefulness of Malaysian Altschul, S. F., Madden, T. L., Schaﬀer, A. A., Zhang, J., actinomycetes as screening sources of natural product- Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped based drug discovery programs, but also may be useful as BLAST and PSI-BLAST: a new generation of protein database basic data such as distribution studies. search programs. Nucleic Acids Res. 25, 3389–3402. DNA Data Bank of Japan. http://www.ddbj.nig.ac.jp/. Acknowledgements Goodfellow, M., Brown, A. B., Cai, J., Chun, J. & Collins, M. D. (1997). Amycolatopsis japonicum sp. nov., an actino- 0 It was a great honor to receive the Hamada Award from mycete producing (S,S)-N,N -ethyenediaminedisuccinic acid. Syst. Appl. Microbiol. 20, 78–84. the Society for Actinomycetes Japan in 2005, in which Ishikawa, J., Tsuchizaki, N., Yoshida, M., Ishiyama, D. & regard I thank the members of the SAJ nomination Hotta, K. (2000). Colony PCR for detection of speciﬁc DNA committee. I am grateful to Dr. Neelam Shahab (SIRIM sequences in actinomycetes. Actinomycetol. 14, 1–5. Berhad, Malaysia) for the collaboration. Special thanks to Keswani, J. & Whitman, W. B. (2001). Relationship of 16S Dr. Motohiro Hino and Mr. Yasuhisa Tsurumi (Astellas rRNA sequence similarity to DNA hybridization in prokaryotes. Pharma Inc.) for providing me with the opportunity to Int. J. Syst. Evol. Microbiol. 51, 667–678.

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