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日 本 I 放 C 線 菌 学 http://www. actino.jp/ 会 日本放線菌学会誌 第28巻 1 号 誌 Published by ACTINOMYCETOLOGICA VOL.28 NO.1, 2014 The Society for Actinomycetes Japan SAJ NEWS

Vol. 31, No. 1, 2017

Contents

• Outline of SAJ: Activities and Membership S 2 • List of new scientific names and nomenclatural changes in the phylum S 3 validly published in 2016 • Award Lecture (Dr. Shumpei Asamizu) S 30 • Publication of Award Lecture (Dr. Shumpei Asamizu) S 41 • Award Lecture (Dr. Takashi Kawasaki) S 42 • Publication of Award Lecture (Dr. Takashi Kawasaki) S 47 • 60th Regular Colloquim S 48 • The 2017 Annual Meeting of the Society for Actinomycetes Japan S 49 • Online access to The Journal of Antibiotics for SAJ members S 50

S1 Outline of SAJ: Activities and Membership

The Society for Actinomycetes Japan (SAJ) membership, please contact the SAJ secretariat was established in 1955 and authorized as a (see below). Annual membership fees are currently scientific organization by Science Council of Japan 5,000 yen for active members, 3,000 yen for in 1985. The Society for Applied Genetics of student members and 20,000 yen or more for Actinomycetes, which was established in 1972, supporting members (mainly companies), merged in SAJ in 1990. SAJ aims at promoting provided that the fees may be changed without actinomycete researches as well as social and advance announcement. scientific exchanges between members domestically and internationally. The Activities of The current members (April 2016 - March 2018) SAJ have included annual and regular scientific of the Board of Directors are: Masayuki Hayakawa meetings, workshops and publications of The (Chairperson; Univ. of Yamanashi), Tohru Dairi Journal of Antibiotics (the official journal, joint (Vice Chairperson; Hokkaido Univ.), Tomohiko publication with Japan Antibiotics Research Tamura (Secretary General; NITE), Takayuki Association), Actinomycetologica (Newsletter) Kajiura (Ajinomoto Co., Inc.), Jun Ishikawa and laboratory manuals. Contributions to (NIID), Hiroyasu Onaka (Tokyo Univ.), Yojiro International Streptomyces Project (ISP) and Anzai (Toho Univ.), Yoshimitsu Hamano (Fukui International Symposium on Biology of Pref. Univ.), Masayuki Igarashi (Institute of Actinomycetes (ISBA) have also been SAJ's Microbial Chemistry), Akira Arisawa activities. In addition, SAJ have occasional special (MicroBiopharm Japan Co., Ltd.), Takuji projects such as the publication of books related to Nakashima (Kitasato Univ.), Masaaki Kizuka actinomycetes: “Atlas of Actinomycetes, 1997”, (Daiichi Sankyo Co., Ltd.), Hisashi Kawasaki “Identification Manual of Actinomycetes, 2001” (Tokyo Denki Univ.), Takuji Kudo (RIKEN) and “Digital Atlas of Actinomycetes, 2002” Atsuko Matsumoto (Kitasato Univ.), Hideki (http://atlas.actino.jp/). These activities have Yamamura (Univ. of Yamanashi), and Hideyuki been planned and organized by the board of Muramatsu (Institute of Microbial Chemistry). directors with association of executive committees The members of the Advisory Board are: Yuzuru consisting of active members who belong to Mikami, Akira Yokota, Hiroyuki Osada, and Keiko academic and nonacademic organizations. Ochiai. The SAJ Memberships comprise active members, student members, supporting Copyright: The copyright of the articles members and honorary members. Currently (as published in Actinomycetologica is transferred of Dec. 31, 2016), SAJ has about 423 active from the authors to the publisher, The Society for members including student members, 22 oversea Actinomycetes Japan, upon acceptance of the members, 11 honorary members, 3 oversea manuscript. honorary members, 1 special member and 13 supporting members. The SAJ members are The SAJ Secretariat allowed to join the scientific and social meetings c/o Culture Collection Division, Biological or projects (regular and specific) of SAJ on a Resource Center, National Institute of membership basis and to browse The Journal of Technology and Evaluation (NBRC) Antibiotics from a link on the SAJ website and will 2-5-8, Kazusakamatari, Kisarazu, receive each issue of Actinomycetologica, Chiba 292-0818, Japan currently published in June and December. Phone: +81-438-20-5763 Actinomycete researchers in foreign countries are Fax: +81-438-52-2329 welcome to join SAJ. For application of SAJ E-mail: [email protected]

S2 List of new scientific names and nomenclatural changes in the phylum Actinobacteria validly published in 2016

NEW ORDER

Egibacterales Zhang et al. 2016, ord. nov. Egicoccales Zhang et al. 2016, ord. nov. Type genus: Egibacter Zhang et al. 2016. Type genus: Egicoccus Zhang et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 283-289. 66: 530-535. A member of the class Nitriliruptoria. A member of the class Nitriliruptoria.

NEW FAMILY

Egibacteraceae Zhang et al. 2016, fam. nov. A member of the order Egicoccales. Type genus: Egibacter Zhang et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Parviterribacteraceae Foesel et al. 2016, fam. 66: 283-289. nov. A member of the order Egibacterales. Type genus: Parviterribacter Foesel et al. 2016. Egicoccaceae Zhang et al. 2016, fam. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type genus: Egicoccus Zhang et al. 2016. 66: 652-665. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the order Solirubrobacterales. 66: 530-535.

NEW GENUS

Acidipropionibacterium Scholz and Kilian Actinorhabdospora Mingma et al. 2016, gen. 2016, gen. nov. nov. Type : Acidipropionibacterium jensenii Type species: Actinorhabdospora filicis (van Niel 1928) Scholz and Kilian 2016. Mingma et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4422-4432. 66: 3071-3077. A member of the family Propionibacteriaceae. A member of the family Micromonosporaceae.

Actinocrispum Hatano et al. 2016, gen. nov. Allohumibacter Kim et al. 2016, gen. nov. Type species: Actinocrispum wychmicini Type species: Allohumibacter endophyticus Hatano et al. 2016. Kim et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4779-4784. 66: 1823-1827. A member of the family Pseudonocardiaceae. A member of the family .

Actinorectispora Quadri et al. 2016, gen. nov. Cnuibacter Zhou et al. 2016, gen. nov. Type species: Actinorectispora indica Quadri et Type species: Cnuibacter physcomitrellae Zhou al. 2016. et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 939-945. 66: 680-688. A member of the family Pseudonocardiaceae. A member of the family Microbacteriaceae.

S3 Cutibacterium Scholz and Kilian 2016, gen. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 2929-2935. Type species: Cutibacterium acnes (Gilchrist A member of the suborder Corynebacterineae. 1900) Scholz and Kilian 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Lipingzhangella Zhang et al. 2016, gen. nov. 66: 4422-4432. Type species: Lipingzhangella halophila Zhang A member of the family Propionibacteriaceae. et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Egibacter Zhang et al. 2016, gen. nov. 66: 4071-4076. Type species: Egibacter rhizosphaerae Zhang A member of the family Nocardiopsaceae. et al. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Monashia Azman et al. 2016, gen. nov. 66: 283-289. Type species: Monashia flava Azman et al. A member of the family Egibacteraceae. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Egicoccus Zhang et al. 2016, gen. nov. 66: 554-561. Type species: Egicoccus halophilus Zhang et al. A member of the family Intrasporangiaceae. 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Paenarthrobacter Busse 2016, gen. nov. 66: 530-535. Type species: Paenarthrobacter aurescens A member of the family Egicoccaceae. (Phillips 1953) Busse 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Enorma Mishra et al. 2016, gen. nov. 66: 9-37. Type species: Enorma massiliensis Mishra et al. A member of the family Micrococcaceae. 2016. Reference: Stand. Genomic Sci., 2013, 8: Paeniglutamicibacter Busse 2016, gen. nov. 290-305; Validation List no. 168 [Int. J. Syst. Type species: Paeniglutamicibacter sulfureus Evol. Microbiol., 2016, 66: 1603-1606]. (Stackebrandt et al. 1984) Busse 2016. A member of the family Coriobacteriaceae. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Glutamicibacter Busse 2016, gen. nov. A member of the family Micrococcaceae. Type species: Glutamicibacter protophormiae (Lysenko 1959) Busse 2016. Parafrigoribacterium Kong et al. 2016, gen. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 9-37. Type species: Parafrigoribacterium A member of the family Micrococcaceae. mesophilum (Dastager et al. 2008) Kong et al. 2016. Herbihabitans Zhang et al. 2016, gen. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type species: Herbihabitans rhizosphaerae 66: 5252-5259. Zhang et al. 2016. A member of the family Microbacteriaceae. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4156-4161. Parviterribacter Foesel et al. 2016, gen. nov. A member of the family Pseudonocardiaceae. Type species: Parviterribacter kavangonensis Foesel et al. 2016. Huakuichenia Zhang et al. 2016, gen. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type species: Huakuichenia soli Zhang et al. 66: 652-665. 2016. A member of the family Parviterribacteraceae. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5399-5405. Populibacterium Li et al. 2016, gen. nov. A member of the family Microbacteriaceae. Type species: Populibacterium corticicola Li et al. 2016. Lawsonella Bell et al. 2016, gen. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type species: Lawsonella clevelandensis Bell 66: 3743-3748. et al. 2016. A member of the family Jonesiaceae.

S4 Pseudarthrobacter Busse 2016, gen. nov. Salilacibacter Li et al. 2016, gen. nov. Type species: Pseudarthrobacter Type species: Salilacibacter albus Li et al. polychromogenes (Schippers-Lammertse et 2016. al. 1963) Busse 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 2558-2565. 66: 9-37. A member of the family Glycomycetaceae. A member of the family Micrococcaceae. Sediminivirga Zhang et al. 2016, gen. nov. Pseudoglutamicibacter Busse 2016, gen. nov. Type species: Sediminivirga luteola Zhang et al. Type species: Pseudoglutamicibacter 2016. cumminsii (Funke et al. 1997) Busse 2016. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1494-1498. 66: 9-37. A member of the family Brevibacteriaceae. A member of the family Micrococcaceae. Sphaerimonospora Mingma et al. 2016, gen. Pseudopropionibacterium Scholz and Kilian nov. 2016, gen. nov. Type species: Sphaerimonospora cavernae Type species: Pseudopropionibacterium Mingma et al. 2016. propionicum (Buchanan and Pine 1962) Reference: Int. J. Syst. Evol. Microbiol., 2016, Scholz and Kilian 2016. 66: 1735-1744. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the family Streptosporangiaceae. 66: 4422-4432. A member of the family Propionibacteriaceae. Timonella Mishra et al. 2016, gen. nov. Type species: Timonella senegalensis Mishra et Raineyella Pikuta et al. 2016, gen. nov. al. 2016. Type species: Raineyella antarctica Pikuta et al. Reference: Stand. Genomic Sci. 8: 318-335; 2016. Validation List no. 170 [Int. J. Syst. Evol. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbiol., 2016, 66: 2463-2466]. 66: 5529-5536. A member of the order . A member of the family Propionibacteriaceae.

NEW SPECIES

Actinocorallia lasiicapitis Liu et al. 2016, sp. 66: 2724-2729. nov. Type strain: strain 3H-GS17 = CGMCC 4.7282 Actinomadura gamaensis Abagana et al. 2016, = DSM 100595 = JCM 31763. sp. nov. Reference: Int. J. Syst. Evol. Micro-biol., 2016, Type strain: strain NEAU-Gz5 = CGMCC 66: 2172-2177. 4.7301 = DSM 100815. Reference: Antonie van Leeuwenhoek, 2016, Actinocrispum wychmicini Hatano et al. 2016, 109: 833-839; Validation List no. 171 [Int. J. sp. nov. Syst. Evol. Microbiol., 2016, 66: Type strain: strain MI503-A4 = DSM 45934 = 3761-3764]. NBRC 109632. Reference: Int. J. Syst. Evol. Microbiol., 2016, Actinomadura jiaoheensis Zhao et al. 2016, sp. 66: 4779-4784. nov. Type strain: strain NEAU-Jh1-3 = CGMCC Actinomadura adrarensis Lahoum et al. 2016, 4.7197 = DSM 102127 = JCM 30341. sp. nov. Reference: Antonie van Leeuwenhoek, 2015, Type strain: strain ACD12 = CECT 8842 = 108: 1331-1339; Validation List no. 168 [Int. DSM 46745 = JCM 31696. J. Syst. Evol. Microbiol., 2016, 66: Reference: Int. J. Syst. Evol. Microbiol., 2016, 1603-1606].

S5 nov. Actinomadura montaniterrae Songsumanus et Type strain: strain YIM 75728 = CCTCC AA al. 2016, sp. nov. 209065 = DSM 45410. Type strain: strain CYP1-1B = JCM 16995 = Reference: Int. J. Syst. Evol. Microbiol., 2016, KCTC 39784 = PCU 349 = TISTR 2400. 66: 939-945. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3310-3316. Actinorhabdospora filicis Mingma et al. 2016, sp. nov. Actinomadura sporangiiformans Zhao et al. Type strain: strain K12-0408 = NBRC 111897 2016, sp. nov. = TBRC 5327. Type strain: strain NEAU-Jh2-5 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 4.7211 = JCM 30342. 66: 3071-3077. Reference: Antonie van Leeuwenhoek, 2015, 108: 1331-1339; Validation List no. 168 [Int. Agromyces aureus Corretto et al. 2016, sp. J. Syst. Evol. Microbiol., 2016, 66: nov. 1603-1606]. Type strain: strain AR33 = DSM 101731 = LMG 29235. Actinophytocola algeriensis Bouznada et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 3749-3754. Type strain: strain MB20 = CECT 8960 = DSM 46746. Agromyces binzhouensis et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., Type strain: strain OAct353 = CGMCC 4.7180 2016, 66: 2760-2765. = DSM 28305 = NRRL B-59115. Reference: Int. J. Syst. Evol. Microbiol., 2016, Actinoplanes bogorensis Nurkanto et al. 2016, 66: 2278-2283. sp. nov. Type strain: strain LIPI11-2-Ac043 = InaCC Agromyces insulae Huang et al. 2016, sp. nov. A522 = NBRC 110975. Type strain: strain CFH S0483 = CCTCC AB Reference: J. Antibiot., 2016, 69: 26-30; 2014301 = JCM 31741 = KCTC 39117. Validation List no. 172 [Int. J. Syst. Evol. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbiol., 2016, 66: 4299-4305]. 66: 2002-2007.

Actinoplanes lichenis Phongsopitanun et al. Allohumibacter endophyticus Kim et al. 2016, 2016, sp. nov. sp. nov. Type strain: strain LDG1-22 = JCM 30485 = Type strain: strain MWE-A11 = JCM 19371 = PCU 344 = TISTR 2343. KCTC 29232. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 468-473. 66: 1823-1827.

Actinoplanes subglobosus Ngaemthao et al. Amycolatopsis albispora Zhang et al. 2016, sp. 2016, sp. nov. nov. Type strain: strain A-T 5400 = BCC 42734 = Type strain: strain WP1 = KCTC 39642 = NBRC 109645 = TBRC 5832. MCCC 1A10745. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4850-4855. 66: 3860-3864.

Actinopolyspora salinaria Duangmal et al. Arthrobacter deserti Hu et al. 2016, sp. nov. 2016, sp. nov. Type strain: strain YIM CS25 = CGMCC Type strain: strain HS05-03 = BCC 51286 = 1.15091 = DSM 29935 = KCTC 39544. NBRC 109078. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 2035-2040. 66: 1660-1665. Arthrobacter echini Lee et al. 2016, sp. nov. Actinorectispora indica Quadri et al. 2016, sp. Type strain: strain AM23 = KACC 18260 =

S6 DSM 29493. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain KWS-1 = DSM 28796 = 66: 1887-1893. JCM 30059 = MTCC 11836. Reference: Int. J. Syst. Evol. Microbiol., 2016, Asanoa endophytica Niemhom et al. 2016, sp. 66: 4705-4710. nov. Type strain: strain BR3-1 = BCC 66355 = Brachybacterium horti Singh et al. 2016, sp. NBRC 110002. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain THG-S15-4 = CCTCC AB 66: 1377-1382. 2015116 = KCTC 39563 = JCM 31742. Reference: Int. J. Syst. Evol. Microbiol., 2016, Bifidobacterium aquikefiri Laureys et al. 2016, 66: 189-195. sp. nov. Type strain: strain R 54638 = CCUG 67145 = Brevibacterium sediminis Chen et al. 2016, sp. LMG 28769. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain FXJ8.269 = CGMCC 66: 1281-1286. 1.15472 = DSM 102229. Reference: Int. J. Syst. Evol. Microbiol., 2016, Bifidobacterium eulemuris Michelini et al. 66: 5268-5274. 2016, sp. nov. Type strain: strain LMM_E3 = DSM 100216 = Catellatospora paridis Jia et al. 2016, sp. nov. JCM 30801. Type strain: strain NEAU-CL2 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 4.7236 = DSM 100519. 66: 1567-1576. Reference: Antonie van Leeuwenhoek, 2016, 109: 43-50; Validation List no. 169 [Int. J. Bifidobacterium hapali Michelini et al. 2016, Syst. Evol. Microbiol., 2016, 66: sp. nov. 1913-1915]. Type strain: strain MRM_8.14 = DSM 100202 = JCM 30799. Catellatospora vulcania Jia et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 255-265. Type strain: strain NEAU-JM1 = CGMCC 4.7174 = JCM 30054. Bifidobacterium myosotis Michelini et al. 2016, Reference: Antonie van Leeuwenhoek, 2016, sp. nov. 109: 43-50; Validation List no. 169 [Int. J. Type strain: strain MRM_5.9 = DSM 100196 = Syst. Evol. Microbiol., 2016, 66: JCM 30796. 1913-1915]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 255-265. Catenulispora fulva Lee and Whang 2016, sp. nov. Bifidobacterium tissieri Michelini et al. 2016, Type strain: strain SA-246 = KACC 17878 = sp. nov. NBRC 110074. Type strain: strain MRM_5.18 = DSM 100201 Reference: Int. J. Syst. Evol. Microbiol., 2016, = JCM 30798. 66: 271-275. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 255-265. Cellulosimicrobium aquatile Sultanpuram et al. 2016, sp. nov. Blastococcus capsensis Hezbri et al. 2016, sp. Type strain: strain 3bp = KCTC 39527 = LMG nov. 28646 = MCC 2761. Type strain: strain BMG 804 = CECT 8876 = Reference: Antonie van Leeuwenhoek, 2015, DSM 46835. 108: 1357-1364; Validation List no. 168 [Int. Reference: Int. J. Syst. Evol. Microbiol., 2016, J.Syst. Evol. Microbiol., 2016, 66: 66: 4864-4872. 1603-1606].

Brachybacterium aquaticum Kaur et al. 2016, Cellulosimicrobium marinum Hamada et al.

S7 2016, sp. nov. LMG 28277. Type strain: strain RS-7-4 = InaCC A726 = Reference: Int. J. Syst. Evol. Microbiol., 2016, NBRC 110994. 66: 2803-2812. Reference: Arch. Microbiol., 2016, 198: 439-444; Validation List no. 171 [Int. J. Syst. Corynebacterium pollutisoli Negi et al. 2016, Evol. Microbiol., 2016, 66: 3761-3764]. sp. nov. Type strain: strain VDS11 = DSM 100104 = Cnuibacter physcomitrellae Zhou et al. 2016, KCTC 39687 = MCC 2722. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain XA = CGMCC 1.15041 = 66: 3531-3537. DSM 29843. Reference: Int. J. Syst. Evol. Microbiol., 2016, Corynebacterium uropygiale Braun et al. 2016, 66: 680-688. sp. nov. Type strain: strain Iso10 = DSM 46817 = LMG Collinsella massiliensis Padmanabhan et al. 28616. 2016, sp. nov. Reference: Syst. Appl. Microbiol., 2016, 39: Type strain: strain GD3 = CSUR P902 = DSM 88-92; Validation List no. 170 [Int. J. Syst. 26110. Evol. Microbiol., 2016, 66: 2463-2466]. Reference: Stand. Genomic Sci., 2014, 9: 1144-1158; Validation List no. 170 [Int. J. Dactylosporangium solaniradicis Fan et al. Syst. Evol. Microbiol., 2016, 66: 2016, sp. nov. 2463-2466]. Type strain: strain NEAU-FJL2 = CGMCC 4.7302 = DSM 100814. Corynebacterium crudilactis Zimmermann et Reference: Antonie van Leeuwenhoek, 2016, al. 2016, sp. nov. 109: 971-977; Validation List no. 171 [Int. J. Type strain: strain JZ16 = CCUG 69192 = Syst. Evol. Microbiol., 2016, 66: DSM 100882 = LMG 29813. 3761-3764]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5288-5293. Dactylosporangium sucinum Phongsopitanun et al. 2016, sp. nov. Corynebacterium faecale Chen et al. 2016, sp. Type strain: strain RY35-23 = JCM 19831 = nov. PCU 333 = TISTR 2212. Type strain: strain YIM 101505 = CCTCC AB Reference: J. Antibiot., 2015, 68: 379-384; 2013226 = DSM 45971 = JCM 31743. Validation List no. 167 [Int. J. Syst. Evol. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbiol., 2016, 66: 1-3]. 66: 2478-2483. Demequina litorisediminis Park et al. 2016, sp. Corynebacterium guangdongense Li et al. nov. 2016, sp. nov. Type strain: strain GHD-1 = KCTC 52260 = Type strain: strain S01 = CCTCC AB 2015423 NBRC 112299. = GDMCC 1.1022 = KCTC 39608. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4197-4203. 66: 3201-3206. Dermabacter jinjuensis Park et al. 2016, sp. Corynebacterium lowii Bernard et al. 2016, sp. nov. nov. Type strain: strain 32 = DSM 101003 = NCCP Type strain: strain R-50085 = CCUG 65815 = 16133. LMG 28276. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 2573-2577. 66: 2803-2812. Dermabacter vaginalis Chang et al. 2016, sp. Corynebacterium oculi Bernard et al. 2016, sp. nov. nov. Type strain: strain AD1-86 = DSM 100050 = Type strain: strain R-50187 = CCUG 65816 = KCTC 39585.

S8 Reference: Int. J. Syst. Evol. Microbiol., 2016, Frankia casuarinae Nouioui et al. 2016, sp. 66: 1881-1886. nov. Type strain: strain CcI3 = CECT 9043 = DSM Egibacter rhizosphaerae Zhang et al. 2016, sp. 45818. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain EGI 80759 = CGMCC 66: 5201-5210. 1.14997 = KCTC 39588. Reference: Int. J. Syst. Evol. Microbiol., 2016, Frankia elaeagni Nouioui et al. 2016, sp. nov. 66: 283-289. Type strain: strain BMG5.12 = CECT 9031 = DSM 46783. Egicoccus halophilus Zhang et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 5201-5210. Type strain: strain EGI 80432 = CGMCC 1.14988 = KCTC 33612. Friedmanniella aerolata Kim et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 530-535. Type strain: strain 7515T-26 = DSM 27139 = KACC 17306. Enorma massiliensis Mishra et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 1970-1975. Type strain: strain phI = CSUR P183 = DSM 25476. Friedmanniella endophytica Tuo et al. 2016, Reference: Stand. Genomic Sci., 2013, 8: sp. nov. 290-305; Validation List no. 168 [Int. J. Syst. Type strain: strain 4Q3S-3 = CGMCC 4.7307 = Evol. Microbiol., 2016, 66: 1603-1606]. DSM 100723. Reference: Int. J. Syst. Evol. Microbiol., 2016, Enorma timonensis Ramasamy et al. 2016, sp. 66: 3057-3062. nov. Type strain: strain GD5 = CSUR P900 = DSM Frigoribacterium salinisoli Kong et al. 2016, 26111. sp. nov. Reference: Stand. Genomic Sci., 2014, 9: Type strain: strain LAM9155 = ACCC 19902 = 970-986; Validation List no. 170 [Int. J. Syst. JCM 30848. Evol. Microbiol., 2016, 66: 2463-2466]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5252-5259. Enterorhabdus muris Lagkouvardos et al. 2016, sp. nov. Geodermatophilus pulveris Hezbri et al. 2016, Type strain: strain WCA-131-CoC-2 = DSM sp. nov. 29508 = KCTC 15543. Type strain: strain BMG 825 = CECT 9003 = Reference: Nat. Microbiol., 2016, 1: 16131; DSM 46839. Validation List no. 172 [Int. J. Syst. Evol. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbiol., 2016, 66: 4299-4305]. 66: 3828-3834.

Flexivirga endophytica Gao et al. 2016, sp. Glycomyces lacisalsi Guan et al. 2016, sp. nov. nov. Type strain: strain XHU 5089 = CCTCC AA Type strain: strain YIM 7505 = CGMCC 2015034 = JCM 31432 = KCTC 39688. 1.15085 = JCM 30628 = KCTC 39536. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5366-5370. 66: 3388-3392. Gordonia didemni de Menezes et al. 2016, sp. Flexivirga lutea Kang et al. 2016, sp. nov. nov. Type strain: strain TBS-100 = JCM 31200 = Type strain: strain B204 = CBMAI 1069 = KCTC 39625. DSM 46679. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Antonie van Leeuwenhoek, 2016, 66: 3594-3599. 109: 297-303; Validation List no. 169 [Int. J. Syst. Evol. Microbiol., 2016, 66:

S9 1913-1915]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3972-3977. Gordonia hongkongensis Tsang et al. 2016, sp. nov. Kibdelosporangium banguiense Pascual et al. Type strain: strain HKU50 = CCOS 955 = CIP 2016, sp. nov. 111027 = JCM 31934 = NBRC 111234 = Type strain: strain F-240,109 = DSM 46670 = NCCP 16210. LMG 28181. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Antonie van Leeuwenhoek, 2016, 66: 3942-3950. 109: 685-695; Validation List no. 170 [Int. J. Syst. Evol. Microbiol., 2016, 66: Hamadaea flava Chu et al. 2016, sp. nov. 2463-2466]. Type strain: strain YIM C0533 = CGMCC 4.7289 = CPCC 204160 = DSM 100517 = Kineococcus mangrovi Duangmal et al. 2016, KCTC 39591. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain L2-1-L1 = BCC 75409 = 66: 1818-1822. NBRC 110933. Reference: Int. J. Syst. Evol. Microbiol., 2016, Herbihabitans rhizosphaerae Zhang et al. 66: 1230-1235. 2016, sp. nov. Type strain: strain CPCC 204279 = DSM Kocuria arsenatis Román-Ponce et al. 2016, sp. 101727 = NBRC 111774. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain CM1E1 = CCBAU 101092 = 66: 4156-4161. HAMBI 3625 = LMG 28671. Reference: Int. J. Syst. Evol. Microbiol., 2016, Hoyosella rhizosphaerae Li et al. 2016, sp. 66: 1027-1033. nov. Type strain: strain J12GA03 = CGMCC Kocuria pelophila Hamada et al. 2016, sp. nov. 1.15478 = DSM 101985. Type strain: strain RS-2-3 = InaCC A704 = Reference: Int. J. Syst. Evol. Microbiol., 2016, NBRC 110990. 66: 4716-4722. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3276-3280. Huakuichenia soli Zhang et al. 2016, sp. nov. Type strain: strain LIP-1 = CCTCC AB Kocuria subflava Jiang et al. 2016, sp. nov. 2015422 = KCTC 39698. Type strain: strain YIM 13062 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 4.7252 = JCM 31771 = KCTC 39547. 66: 5399-5405. Reference: Antonie van Leeuwenhoek, 2015, 108: 1349-1355; Validation List no. 170 [Int. Humibacter soli Park et al. 2016, sp. nov. J. Syst. Evol. Microbiol., 2016, 66: Type strain: strain R1-20 = JCM 31015 = 2463-2466]. KCTC 39614. Reference: Int. J. Syst. Evol. Microbiol., 2016, Kribbella pittospori Kaewkla et al. 2016, sp. 66: 2509-2514. nov. Type strain: strain PIP 158 = DSM 23717 = Isoptericola cucumis Kämpfer et al. 2016, sp. NRRL B-24813. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain AP-3 = CCM 86538 = DSM 66: 2284-2290. 101603 = JCM 31758 = LMG 29223. Reference: Int. J. Syst. Evol. Microbiol., 2016, Lawsonella clevelandensis Bell et al. 2016, sp. 66: 2784-2788. nov. Type strain: strain X1036 = CCUG 66657 = Jatrophihabitans huperziae Gong et al. 2016, DSM 45743. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain CPCC 204076 = I13A-01604 66: 2929-2935. = DSM 46866 = NBRC 110718.

S10 Lentzea guizhouensis Cao et al. 2016, sp. nov. 66: 4665-4670. Type strain: strain DHS C013 = CGMCC 4.7203 = DSM 102208 = KCTC 29677. Microbacterium diaminobutyricum Fidalgo et Reference: Antonie van Leeuwenhoek, 2015, al. 2016, sp. nov. 108: 1365-1372; Validation List no. 168 [Int. Type strain: strain RZ63 = CECT 8355 = DSM J. Syst. Evol. Microbiol., 2016, 66: 27101. 1603-1606]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4492-4500. Leucobacter holotrichiae Zhu et al. 2016, sp. nov. Microbacterium faecale Chen et al. 2016, sp. Type strain: strain T14 = DSM 28968 = JCM nov. 30245. Type strain: strain YIM 101168 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 1.15152 = DSM 27232 = KCTC 39554. 66: 1857-1861. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4445-4450. Leucobacter populi Fang et al. 2016, sp. nov. Type strain: strain 06C10-3-11 = CFCC 12199 Microbacterium gilvum Chen et al. 2016, sp. = KCTC 39685. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain YIM 100951 = CCTCC AB 66: 2254-2258. 2012971 = DSM 26235 = JCM 18537. Reference: Antonie van Leeuwenhoek, 2016, Lipingzhangella halophila Zhang et al. 2016, 109: 1177-1183; Validation List no. 172 [Int. sp. nov. J. Syst. Evol. Microbiol., 2016, 66: Type strain: strain EGI 80537 = CGMCC 4299-4305]. 4.7224 = DSM 102030. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbacterium sorbitolivorans Meng et al. 66: 4071-4076. 2016, sp. nov. Type strain: strain SZDIS-1-1 = CGMCC Mariniluteicoccus endophyticus Liu et al. 1.15228 = DSM 103422. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain YIM 2617 = KCTC 29482 = 66: 5556-5561. DSM 28728 = JCM 30097. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbispora camponoti Han et al. 2016, sp. 66: 1306-1310. nov. Type strain: strain 2C-HV3 = CGMCC 4.7281 Marmoricola ginsengisoli Lee et al. 2016, sp. = DSM 100527 = JCM 31773. nov. Reference: Antonie van Leeuwenhoek, 2016, Type strain: strain Gsoil 097 = DSM 22772 = 109: 215-223; Validation List no. 169 [Int. J. KACC 14267. Syst. Evol. Microbiol., 2016, 66: Reference: Int. J. Syst. Evol. Microbiol., 2016, 1913-1915]. 66: 1996-2001. Microlunatus endophyticus Tuo et al. 2016, sp. Marmoricola pocheonensis Lee et al. 2016, sp. nov. nov. Type strain: strain S3Af-1 = CGMCC 4.7306 = Type strain: strain Gsoil 818 = DSM 22773 = DSM 100019 = JCM 31774. KACC 14275. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 481-486. 66: 1996-2001. Microlunatus nigridraconis Zhang et al. 2016, Microbacterium aureliae Kaur et al. 2016, sp. sp. nov. nov. Type strain: strain CPCC 203993 = DSM Type strain: strain JF-6 = JCM 30060 = KCTC 29529 = KCTC 29689 = NBRC 110715. 39828 = MTCC 11843. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3614-3618.

S11 Micromonospora vinacea Carro et al. 2016, sp. Micromonospora mangrovi Xie et al. 2016, sp. nov. nov. Type strain: strain GUI63 = CECT 9019 = Type strain: strain 2803GPT1-18 = CCTCC AA DSM 101695. 2012012 = DSM 45761. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Antonie van Leeuwenhoek, 2016, 66: 3509-3514. 109: 483-498; Validation List no. 170 [Int. J. Syst. Evol. Microbiol., 2016, 66: Micromonospora vulcania Jia et al. 2016, sp. 2463-2466]. nov. Type strain: strain NEAU-JM2 = CGMCC Micromonospora noduli Carro et al. 2016, sp. 4.7144 = DSM 46711 = JCM 31777. nov. Reference: Antonie van Leeuwenhoek, 2015, Type strain: strain GUI43 = CECT 9020 = 108: 1383-1390; Validation List no. 168 [Int. DSM 101694. J. Syst. Evol. Microbiol., 2016, 66: Reference: Int. J. Syst. Evol. Microbiol., 2016, 1603-1606]. 66: 3509-3514. Monashia flava Azman et al. 2016, sp. nov. Micromonospora ovatispora Li and Hong 2016, Type strain: strain MUSC 78 = DSM 29621 = sp. nov. MCCC 1K00454 = NBRC 110749. Type strain: strain 2701SIM06 = CCTCC AA Reference: Int. J. Syst. Evol. Microbiol., 2016, 2012009 = DSM 45759. 66: 554-561. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 889-893. Mumia xiangluensis Zhou et al. 2016, sp. nov. Type strain: strain NEAU-KD1 = CGMCC Micromonospora profundi Veyisoglu et al. 4.7305 = DSM 101040. 2016, sp. nov. Reference: Antonie van Leeuwenhoek, 2016, Type strain: strain DS3010 = DSM 45981 = 109: 827-832; Validation List no. 171 [Int. J. KCTC 29243. Syst. Evol. Microbiol., 2016, 66: Reference: Int. J. Syst. Evol. Microbiol., 2016, 3761-3764]. 66: 4735-4743. Mycobacterium alsense Tortoli et al. 2016, sp. Micromonospora sediminis Phongsopitanun et nov. al. 2016, sp. nov. Type strain: strain TB 1906 = CCUG 56586 = Type strain: strain CH3-3 = JCM 18523 = PCU DSM 45230. 350 = TISTR 2396. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 450-456. 66: 3235-3240. Mycobacterium arcueilense Konjek et al. 2016, Micromonospora soli Thawai et al. 2016, sp. sp. nov. nov. Type strain: strain 269 = ParisRGMnew_3 = Type strain: strain SL3-70 = BCC 67268 = CIP 110654 = DSM 46715. NBRC 110009. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Antonie van Leeuwenhoek, 2016, 66: 3694-3702. 109: 449-456; Validation List no. 170 [Int. J. Syst. Evol. Microbiol., 2016, 66: Mycobacterium helvum Tran and Dahl 2016, 2463-2466]. sp. nov. Type strain: strain DL739 = JCM 30396 = Micromonospora ureilytica Carro et al. 2016, NCCB 100520. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain GUI23 = CECT 9022 = 66: 4480-4485. DSM 101692. Reference: Int. J. Syst. Evol. Microbiol., 2016, Mycobacterium lutetiense Konjek et al. 2016, 66: 3509-3514. sp. nov. Type strain: strain 071 = ParisRGMnew_1 =

S12 CIP 110656 = DSM 46713. Nesterenkonia aurantiaca Finore et al. 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 3694-3702. Type strain: strain CK5 = DSM 27373 = JCM 19723. Mycobacterium montmartrense Konjek et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 1554-1560. Type strain: strain 196 = ParisRGMnew_2 = CIP 110655 = DSM 46714. Nesterenkonia massiliensis Edouard et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 3694-3702. Type strain: strain NP1 = CSUR P244 = DSM 26221. Mycobacterium oryzae Ramaprasad et al. 2016, Reference: Stand. Genomic Sci., 2014, 9: sp. nov. 866-882; Validation List no. 170 [Int. J. Syst. Type strain: strain JC290 = KCTC 39560 = Evol. Microbiol., 2016, 66: 2463-2466]. LMG 28809. Reference: Int. J. Syst. Evol. Microbiol., 2016, Nocardia camponoti Liu et al. 2016, sp. nov. 66: 4530-4536. Type strain: strain 1H-HV4 = CGMCC 4.7278 = DSM 100526. Mycobacterium paraintracellulare Lee et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 1900-1905. Type strain: strain MOTT64 = KCTC 29084 = JCM 30622. Nocardia jiangsuensis Bai et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain KLBMP S0027 = CGMCC 66: 3132-3141. 4.7330 = DSM 101725 = KCTC 39691. Reference: Int. J. Syst. Evol. Microbiol., 2016, Mycobacterium paraterrae Lee et al. 2016, sp. 66: 4633-4638. nov. Type strain: strain 05-2522 = DSM 45127 = Nocardia rayongensis Tanasupawat et al. 2016, KCTC 19556. sp. nov. Reference: Microbiol. Immunol. 2010, 54: Type strain: strain RY45-3 = JCM 19832 = 46-53; Validation List no. 172 [Int. J. Syst. PCU 334 = TISTR 2213. Evol. Microbiol., 2016, 66: 4299-4305]. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1950-1955. Mycobacterium sarraceniae Tran and Dahl 2016, sp. nov. Nocardia shinanonensis Matsumoto et al. Type strain: strain DL734 = JCM 30395 = 2016, sp. nov. NCCB 100519. Type strain: strain IFM 11456 = NBRC 109590 Reference: Int. J. Syst. Evol. Microbiol., 2016, = TBRC 5149. 66: 4480-4485. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3324-3328. Nakamurella endophytica Tuo et al. 2016, sp. nov. Nocardia zapadnayensis Ozdemir-Kocak et al. Type strain: strain 2Q3S-4-2 = CGMCC 4.7308 2016, sp. nov. = DSM 100722. Type strain: strain FMN18 = DSM 45872 = Reference: Int. J. Syst. Evol. Microbiol., 2016, KCTC 29234. 66: 1577-1582. Reference: Antonie van Leeuwenhoek, 2016, 109: 95-103; Validation List no. 169 [Int. J. Nakamurella silvestris França et al. 2016, sp. Syst. Evol. Microbiol., 2016, 66: nov. 1913-1915]. Type strain: strain S20-107 = DSM 102309 = LMG 29427. Nocardioides albidus Singh et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 5460-5464. Type strain: strain THG-S11.7 = CCTCC AB 2015297 = JCM 31749 = KCTC 39607.

S13 Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1932-1936. 66: 371-378. Nocardioides zeicaulis Kämpfer et al. 2016, sp. Nocardioides baekrokdamisoli Lee et al. 2016, nov. sp. nov. Type strain: strain JM-601 = CCM 8654 = CIP Type strain: strain B2-12 = DSM 100725 = 110980 = DSM 101604. KCTC 39748 = NRRL B-65313. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1869-1874. 66: 4231-4235. Nocardiopsis akesuensis Gao et al. 2016, sp. Nocardioides flavus Wang et al. 2016, sp. nov. nov. Type strain: strain Y4 = CGMCC 1.12791 = Type strain: strain TRM 46250 = CCTCC AA JCM 19770 = LMG 28100 = MCCC 2015027 = KCTC 39725. 1A09944. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5005-5009. 66: 5275-5280. Nocardiopsis ansamitocini Zhang et al. 2016, Nocardioides ginkgobilobae Xu et al. 2016, sp. sp. nov. nov. Type strain: strain EGI 80425 = CGMCC 9969 Type strain: strain SYP-A7303 = DSM 100492 = DSM 103990 = KCTC 39605. = JCM 30556 = KCTC 39594. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 230-235. 66: 2013-2018. Nocardiopsis mwathae Akhwale et al. 2016, sp. Nocardioides intraradicalis Huang et al. 2016, nov. sp. nov. Type strain: strain No.156 = CECT 8552 = Type strain: strain YIM DR1091 = CGMCC DSM 46659. 4.7251 = JCM 30632. Reference: Antonie van Leeuwenhoek, 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 109: 421-430; Validation List no. 170 [Int. J. 66: 3841-3847. Syst. Evol. Microbiol., 2016, 66: 2463-2466]. Nocardioides massiliensis Dubourg et al. 2016, sp. nov. Nocardiopsis rhizosphaerae Zhang et al. 2016, Type strain: strain GD13 = CSUR P894 = DSM sp. nov. 28216. Type strain: strain EGI 80674 = CGMCC Reference: New Microbes New Infect., 2016, 4.7228 = DSM 101528 = KCTC 39673. 10: 47-57; Validation List no. 172 [Int. J. Reference: Int. J. Syst. Evol. Microbiol., 2016, Syst. Evol. Microbiol., 2016, 66: 66: 5129-5133. 4299-4305]. Nocardiopsis sediminis Muangham et al. 2016, Nocardioides pakistanensis Amin et al. 2016, sp. nov. sp. nov. Type strain: strain 1SS5-02 = BCC 75410 = Type strain: strain NCCP-1340 = DSM 29942 = NBRC 110934. JCM 30630. Reference: Int. J. Syst. Evol. Microbiol., Reference: Antonie van Leeuwenhoek, 2016, 2016, 66: 3835-3840. 109: 1101-1109; Validation List no. 172 [Int. J. Syst. Evol. Microbiol., 2016, 66: Nonomuraea gerenzanensis Dalmastri et al. 4299-4305]. 2016, sp. nov. Type strain: strain ATCC 39727 = DSM Nocardioides rotundus Wang et al. 2016, sp. 100948. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain GY0594 = MCCC 1A10561 66: 912-921. = KCTC 39638. Reference: Int. J. Syst. Evol. Microbiol., 2016, Nonomuraea indica Quadri et al. 2016, sp.

S14 nov. Type strain: strain DRQ-2 = CCTCC AA Phycicoccus endophyticus Liu et al. 2016, sp. 209050 = DSM 103467 = JCM 31750 = nov. NCIM 5480. Type strain: strain IP6SC6 = CGMCC 4.7300 = Reference: J. Antibiot., 2015, 68: 491-495; DSM 100020 = JCM 31784. Validation List no. 167 [Int. J. Syst. Evol. Reference: Int. J. Syst. Evol. Microbiol., 2016, Microbiol., 2016, 66: 1-3]. 66: 1105-1111.

Nonomuraea purpurea Suksaard et al. 2016, Phycicoccus ginsengisoli Kang et al. 2016, sp. sp. nov. nov. Type strain: strain 1SM4-01 = BCC 60397 = Type strain: strain DCY87 = JCM 31016 = NBRC 109647. KCTC 39635. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4987-4992. 66: 5320-5327.

Nonomuraea thermotolerans Wu and Liu 2016, Phytoactinopolyspora alkaliphila Zhang et al. sp. nov. 2016, sp. nov. Type strain: strain 3-33-9B = ATCC BAA-2629 Type strain: strain EGI 80629 = CGMCC = CGMCC 4.7161. 4.7225 = DSM 101529 = KCTC 39701. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 894-900. 66: 2058-2063.

Nonomuraea zeae Shen et al. 2016, sp. nov. Phytohabitans kaempferiae Niemhom et al. Type strain: strain NEAU-ND5 = CGMCC 2016, sp. nov. 4.7280 = DSM 100528. Type strain: strain KK1-3 = BCC 66360 = Reference: Int. J. Syst. Evol. Microbiol., 2016, NBRC 110005. 66: 2259-2264. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 2917-2922. Ornithinicoccus halotolerans Zhang et al. 2016, sp. nov. Phytomonospora cypria Sahin et al. 2016, sp. Type strain: strain EGI 80423 = CGMCC nov. 1.14989 = JCM 31779 = KCTC 39700. Type strain: strain KT1403 = DSM 46767 = Reference: Int. J. Syst. Evol. Microbiol., 2016, KCTC 29479. 66: 1894-1899. Reference: Antonie van Leeuwenhoek, 2015, 108: 1425-1432; Validation List no. 169 [Int. Parviterribacter kavangonensis Foesel et al. J. Syst. Evol. Microbiol., 2016, 66: 2016, sp. nov. 1913-1915]. Type strain: strain D16/0/H6 = DSM 25205 = LMG 26950. Planomonospora corallina Suriyachadkun et Reference: Int. J. Syst. Evol. Microbiol., 2016, al. 2016, sp. nov. 66: 652-665. Type strain: strain A-T 11038 = BCC 67829 = NBRC 110609 = TBRC 4489. Parviterribacter multiflagellatus Foesel et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 3224-3229. Type strain: strain A22/0/F9_1 = DSM 25204 = LMG 26949. Plantactinospora soyae Guo et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 652-665. Type strain: strain NEAU-gxj3 = CGMCC 4.7221 = DSM 46832. Patulibacter brassicae Jin et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain SD = CICC 24108 = KCTC 66: 2578-2584. 39817. Reference: Int. J. Syst. Evol. Microbiol., 2016, Populibacterium corticicola Li et al. 2016, sp. 66: 5056-5060. nov.

S15 Type strain: strain 2D-4 = CFCC 11886 = KCTC 33576. Saccharomonospora xiaoerkulensis Li et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 3743-3748. Type strain: strain TRM 41495 = CCTCC AA 2015038 = KCTC 39727. Promicromonospora alba Guo et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 5145-5149. Type strain: strain 1C-HV12 = CGMCC 4.7283 = DSM 100490 = JCM 31782. Saccharopolyspora subtropica Wu et al. 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 1340-1345. Type strain: strain T3 = CGMCC 4.7206 = DSM 46801. Propionibacterium namnetense Aubin et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 1990-1995. Type strain: strain NTS 31307302 = CCUG 66358 = DSM 29427. Saccharothrix isguenensis Bouznada et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 3393-3399. Type strain: strain MB27 = CECT 9045 = DSM 46885. Pseudoclavibacter endophyticus Li et al. 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 4785-4790. Type strain: strain EGI 60007 = CGMCC 1.15081 = DSM 29943 = KCTC 39112 = Saccharothrix lopnurensis Li et al. 2016, sp. JCM 30633. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain YIM LPA2h = CGMCC 66: 1287-1292. 4.7246 = DSM 46881 = JCM 30635 = KCTC 39545. Raineyella antarctica Pikuta et al. 2016, sp. Reference: Antonie van Leeuwenhoek, 2015, nov. 108: 975-981; Validation List no. 167 [Int. J. Type strain: strain LZ-22 = ATCC TSD-18 = Syst. Evol. Microbiol., 2016, 66: 1-3]. DSM 100494 = JCM 30886. Reference: Int. J. Syst. Evol. Microbiol., 2016, Saccharothrix stipae Lin et al. 2016, sp. nov. 66: 5529-5536. Type strain: strain D34 = ACCC 19714 = JCM 30560. Rhodococcus humicola Nguyen and Kim 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 1017-1021. Type strain: strain UC33 = KACC 18500 = NBRC 111581. Salilacibacter albus Li et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain J11Y309 = CGMCC 4.7242 66: 2362-2369. = DSM 46875 = LMG 29297. Reference: Int. J. Syst. Evol. Microbiol., 2016, Rhodococcus pedocola Nguyen and Kim 2016, 66: 2558-2565. sp. nov. Type strain: strain UC12 = KACC 18499 = Sediminivirga luteola Zhang et al. 2016, sp. NBRC 111580. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain F23 = CGMCC 1.12785 = 66: 2362-2369. JCM 19771 = MCCC 1A09945. Reference: Int. J. Syst. Evol. Microbiol., 2016, Rothia aerolata Kämpfer et al. 2016, sp. nov. 66: 1494-1498. Type strain: strain 140917-MRSA-09 = CCM 8669 = DSM 102816 = JCM 31759 = LMG Sinomonas halotolerans Guo et al. 2016, sp. 29446. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain CFH S0499 = CCTCC 66: 3102-3107. AB2014300 = JCM 31751 = KCTC 39116.

S16 Reference: Antonie van Leeuwenhoek, 2015, Streptomyces arcticus Zhang et al. 2016, sp. 108: 887-895; Validation List no. 167 [Int. J. nov. Syst. Evol. Microbiol., 2016, 66: 1-3]. Type strain: strain ZLN234 = CCTCC AA 2015005 = DSM 100713. Sphaerimonospora cavernae Mingma et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 1482-1487. Type strain: strain N74 = BCC 77604 = NBRC 111481. Streptomyces bambusae Nguyen and Kim Reference: Int. J. Syst. Evol. Microbiol., 2016, 2016, sp. nov. 66: 1735-1744. Type strain: strain T110 = KACC 18225 = KEMB 9005-214 = NBRC 110903. Stackebrandtia cavernae Zhang et al. 2016, sp. Reference: Curr. Microbiol., 2015, 71: nov. 658-668; Validation List no. 168 [Int. J. Syst. Type strain: strain YIM ART06 = CCTCC AA Evol. Microbiol., 2016, 66: 1603-1606]. 2015021 = DSM 100594 = KCTC 39599. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces bryophytorum Li et al. 2016, sp. 66: 1206-1211. nov. Type strain: strain NEAU-HZ10 = CGMCC Streptomonospora tuzyakensis Tatar et al. 4.7151 = DSM 42138. 2016, sp. nov. Reference: Antonie van Leeuwenhoek, 2016, Type strain: strain BN506 = DSM 45930 = 109: 1209-1215; Validation List no. 172 [Int. KCTC 29210. J. Syst. Evol. Microbiol., 2016, 66: Reference: Antonie van Leeuwenhoek, 2016, 4299-4305]. 109: 35-41; Validation List no. 169 [Int. J. Syst. Evol. Microbiol., 2016, 66: Streptomyces camponoticapitis Li et al. 2016, 1913-1915]. sp. nov. Type strain: strain 2H-TWYE14 = CGMCC Streptomyces actinomycinicus Tanasupawat et 4.7275 = DSM 100523. al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain RCU-197 = JCM 30864 = 66: 3855-3859. PCU 342 = TISTR 2208. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces canalis Xie et al. 2016, sp. nov. 66: 290-295. Type strain: strain TRM 46794-61 = CCTCC AA 2015006 = DSM 104041 = KCTC 39568. Streptomyces adustus Lee and Wang 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., nov. 2016, 66: 3219-3223. Type strain: strain WH-9 = KACC 17197 = NBRC 109810. Streptomyces chitinivorans Ray et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 3573-3578. Type strain: strain RC1832 = JCM 30611 = KCTC 29696. Streptomyces alfalfae She et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain XY25 = KCTC 39571 = 66: 3241-3248. CCTCC AA2015019 = DSM 103384. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces daqingensis Pan et al. 2016, sp. 66: 44-49. nov. Type strain: strain NEAU-ZJC8 = CGMCC Streptomyces andamanensis Sripreechasak et 4.7178 = JCM 30057. al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain KC-112 = KCTC 29502 = 66: 1358-1363. NBRC 110085 = PCU 347 = TISTR 2401. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces formicae Bai et al. 2016, sp. nov. 66: 2030-2034. Type strain: strain 1H-GS9 = CGMCC 4.7277 = DSM 100524.

S17 Reference: Antonie van Leeuwenhoek, 2016, 109: 225-235; Validation List no. 169 [Int. J. 109: 253-261; Validation List no. 169 [Int. J. Syst. Evol. Microbiol., 2016, 66: Syst. Evol. Microbiol., 2016, 66: 1913-1915]. 1913-1915]. Streptomyces oryzae Mingma et al. 2016, sp. Streptomyces fractus Rohland and Meyers nov. 2016, sp. nov. Type strain: strain S16-07 = BCC 60400 = Type strain: strain MV32 = DSM 42163 = NBRC 109761. NRRL B-59159. Reference: J. Antibiot., 2015, 68: 368-372; Reference: Antonie van Leeuwenhoek, 2015, Validation List no. 168 [Int. J. Syst. Evol. 107: 1127-1134; Validation List no. 168 [Int. Microbiol., 2016, 66: 1603-1606]. J. Syst. Evol. Microbiol., 2016, 66: 1603-1606]. Streptomyces ovatisporus Veyisoglu et al. 2016, sp. nov. Streptomyces hyaluromycini Harunari et al. Type strain: strain S4702 = CGMCC 4.7357 = 2016, sp. nov. DSM 42103 = KCTC 29206. Type strain: strain MB-PO13 = DSM 100105 = Reference: Int. J. Syst. Evol. Microbiol., 2016, NBRC 110483. 66: 4856-4863. Reference: J. Antibiot., 2016, 69: 159-163; Validation List no. 170 [Int. J. Syst. Evol. Streptomyces palmae Sujarit et al. 2016, sp. Microbiol., 2016, 66: 2463-2466]. nov. Type strain: strain CMU-AB204 = JCM 31289 Streptomyces indoligenes Luo et al. 2016, sp. = TBRC 1999. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain TRM 43006 = CCTCC AA 66: 3983-3988. 2015010 = DSM 104005 = KCTC 39611. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces phyllanthi Klykleung et al. 2016, 66: 2424-2428. sp. nov. Type strain: strain PA1-07 = JCM 30865 = Streptomyces kronopolitis Liu et al. 2016, sp. KCTC 39785 = TISTR 2346. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain NEAU-ML8 = CGMCC 66: 3923-3928. 4.7323 = DSM 101986. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces pini Madhaiyan et al. 2016, sp. 66: 5352-5357. nov. Type strain: strain PL19 = ICMP 17783 = Streptomyces lacrimifluminis Zhang et al. NRRL B-24728. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain Z1027 = CGMCC 4.7272 = 66: 4204-4210. JCM 31054. Reference: Int. J. Syst. Evol. Microbiol., 2016, Streptomyces polygonati Guo et al. 2016, sp. 66: 4981-4986. nov. Type strain: strain NEAU-G9 = CGMCC Streptomyces litoralis Ma et al. 2016, sp. nov. 4.7237 = DSM 100521. Type strain: strain TRM 46515 = CCTCC AA Reference: Int. J. Syst. Evol. Microbiol., 2016, 2015040 = KCTC 39729. 66: 1488-1493. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5051-5055. Streptomyces rhizosphaerihabitans Lee and Wang 2016, sp. nov. Streptomyces lonarensis Sharma et al. 2016, sp. Type strain: Stain JR-35 = KACC 17181 = nov. NBRC 109807. Type strain: strain NCL 716 = DSM 42084 = Reference: Int. J. Syst. Evol. Microbiol., 2016, KCTC 39684 = MTCC 11708. 66: 3573-3578. Reference: Antonie van Leeuwenhoek, 2016,

S18 Streptomyces siamensis Sripreechasak et al. Type strain: strain SG1 = CECT 8961 = DSM 2016, sp. nov. 46887. Type strain: strain KC-038 = JCM 18409 = Reference: Int. J. Syst. Evol. Microbiol., 2016, NBRC 108799 = PCU 328 = TISTR 2107. 66: 2484-2490. Reference: J. Antibiot., 2013, 66: 633-640; Validation List no. 168 [Int. J. Syst. Evol. Streptosporangium corydalis Fang et al. 2016, Microbiol., 2016, 66: 1603-1606]. sp. nov. Type strain: strain NEAU-Y6 = CGMCC Streptomyces similanensis Sripreechasak et al. 4.7150 = DSM 46722. 2016, sp. nov. Reference: Antonie van Leeuwenhoek, 2016, Type strain: strain KC-106 = JCM 18410 = 109: 439-448; Validation List no. 170 [Int. J. NBRC 108798 = PCU 329 = TISTR 2104. Syst. Evol. Microbiol., 2016, 66: Reference: J. Antibiot., 2013, 66: 633-640; 2463-2466]. Validation List no. 168 [Int. J. Syst. Evol. Microbiol., 2016, 66: 1603-1606]. Streptosporangium fenghuangense Fang et al. 2016, sp. nov. Streptomyces tremellae Wen et al. 2016, sp. Type strain: strain NEAU-hd-3 = CGMCC nov. 4.7212 = JCM 30058. Type strain: strain Js-1 = CCTCC M 2011365 = Reference: Antonie van Leeuwenhoek, 2016, JCM 30846. 109: 439-448; Validation List no. 170 [Int. J. Reference: Int. J. Syst. Evol. Microbiol., 2016, Syst. Evol. Microbiol., 2016, 66: 66: 5028-5033. 2463-2466].

Streptomyces verrucosisporus Phongsopitanun Streptosporangium jiaoheense Zhao et al. et al. 2016, sp. nov. 2016, sp. nov. Type strain: strain CPB1-1 = CM 18519 = PCU Type strain: strain NEAU-Jh1-4 = CGMCC 343 = TISTR 2344. 4.7213 = JCM 30348. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 3607-3613. 66: 2370-2376.

Streptomyces yangpuensis Tang et al. 2016, sp. Streptosporangium lutulentum Fang et al. nov. 2016, sp. nov. Type strain: strain fd2-tb = CGMCC 4.7256 = Type strain: strain NEAU-FHSN1 = CGMCC DSM 100336. 4.7141 = DSM 46740. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Antonie van Leeuwenhoek, 2016, 66: 1224-1229. 109: 439-448; Validation List no. 170 [Int. J. Syst. Evol. Microbiol., 2016, 66: Streptomyes spongiicola Huang et al. 2016, sp. 2463-2466]. nov. Type strain: strain HNM0071 = Streptosporangium saharense Chaouch et al. CCTCCAA2015018 = DSM 103383 = 2016, sp. nov. KCTC 39604. Type strain: strain SG20 = CECT 8840 = DSM Reference: Int. J. Syst. Evol. Microbiol., 2016, 46743. 66: 738-743. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1371-1376. Streptosporangium algeriense Boubetra et al. 2016, sp. nov. Streptosporangium shengliense Zhang et al. Type strain: strain 169 = CCUG 62974 = DSM 2016, sp. nov. 45455 = MTCC 11561. Type strain: strain NEAU-GH7 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 4.7105 = DSM 45881. 66: 1034-1038. Reference: Antonie van Leeuwenhoek, 2014, 105: 237-243; Antonie van Leeuwenhoek, Streptosporangium becharense Chaabane 2014, 105: 265 (erratum); Validation List no. Chaouch et al. 2016, sp. nov. 168 [Int. J. Syst. Evol. Microbiol., 2016, 66:

S19 1603-1606]. Type strain: strain HKU52 = DSM 100208 = JCM 30715. Streptosporangium taraxaci Zhao et al. 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 391-397. Type strain: strain NEAU-Wp2-0 = CGMCC 4.7217 = JCM 30349. Tsukamurella serpentis Tang et al. 2016, sp. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 2370-2376. Type strain: strain HKU54 = DSM 100915 = JCM 31017. Tenggerimyces flavus Li et al. 2016, sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain S6R2A4-9 = CGMCC 66: 3329-3336. 4.7241 = DSM 28944. Reference: Int. J. Syst. Evol. Microbiol., 2016, Tsukamurella sinensis Teng et al. 2016, sp. 66: 1499-1505. nov. Type strain: strain HKU51 = DSM 100207 = Tersicoccus solisilvae Sultanpuram et al. 2016, JCM 30714. sp. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Type strain: strain 36A = CGMCC 1.15480 = 66: 391-397. KCTC 33776. Reference: Int. J. Syst. Evol. Microbiol., 2016, Umezawaea endophytica Chu et al. 2016, sp. 66: 5061-5065. nov. Type strain: strain YIM 2047X = CPCC 204132 Tessaracoccus flavus Kumari et al. 2016, sp. = DSM 103496 = JCM 30637 = KCTC nov. 39538. Type strain: strain RP1 = DSM 100159 = Reference: Antonie van Leeuwenhoek, 2015, KCTC 39686 = MCC 2769. 108: 667-672; Validation List no. 167 [Int. J. Reference: Int. J. Syst. Evol. Microbiol., 2016, Syst. Evol. Microbiol., 2016, 66: 1-3]. 66: 1862-1868. Verrucosispora sonchi Ma et al. 2016, sp. nov. Tessaracoccus rhinocerotis Li et al. 2016, sp. Type strain: strain NEAU-QY3 = CGMCC nov. 4.7312 = DSM 101530. Type strain: strain YIM 101269 = CCTCC AB Reference: Int. J. Syst. Evol. Microbiol., 2016, 2013217 = DSM 27579. 66: 5430-5436. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 922-927. Williamsia herbipolensis Kämpfer et al. 2016, sp. nov. Timonella senegalensis Mishra et al. 2016, sp. Type strain: strain ARP1 = DSM 46872 = LMG nov. 28679. Type strain: strain JC301 = CSUR P167 = Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 25696. 66: 4609-4613. Reference: Stand. Genomic Sci. 8: 318-335; Validation List no. 170 [Int. J. Syst. Evol. Yimella radicis Yang et al. 2016, sp. nov. Microbiol., 2016, 66: 2463-2466]. Type strain: strain py1292 = DSM 100721 = KCTC 39612 = LMG 29070. Tsukamurella hongkongensis Teng et al. 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, sp. nov. 66: 4191-4196.

NEW SUBSPECIES

Clavibacter michiganensis subsp. capsici Oh Reference: Int. J. Syst. Evol. Microbiol., 2016, et al. 2016, subsp. nov. 66: 4065-4070. Type strain: strain PF008 = KACC 18448 = LMG 29047. Propionibacterium acnes subsp. acnes

S20 (Gilchrist 1900) McDowell et al. 2016, (Gilchrist 1900) Dekio et al. 2015 (Int. J. subsp. nov. Syst. Evol. Microbiol., 2015, 65: 4776-4787) Type strain: strain ATCC 6919 = BCRC 10723 has priority. = CCUG 1794 = CECT 5684 = CGMCC 1.5003 = CIP 53.117 = DSM 1897 = JCM Propionibacterium acnes subsp. defendens 6425 = KCTC 3314 = LMG 16711 = NBRC McDowell et al. 2016, subsp. nov. 107605 = NCTC 737 = NRRL B-4224 = Type strain: strain ATCC 11828 = BCRC 16146 VKM Ac-1450. = CCUG 6369 = JCM 6473 = KCTC 3320. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5358-5365. 66: 5358-5365. Note: Propionibacterium acnes subsp. acnes

NEW COMBINATION

Acidipropionibacterium acidipropionici 66: 4422-4432. (Orla-Jensen 1909) Scholz and Kilian 2016, comb. nov. Acidipropionibacterium olivae (Lucena-Padrós Basonym: Propionibacterium acidipropionici et al. 2014) Scholz and Kilian 2016, comb. Orla-Jensen 1909. nov. Type strain: strain ATCC 25562 = CGMCC Basonym: Propionibacterium olivae 1.2230 = CIP 103025 = DSM 4900 = NBRC Lucena-Padrós et al. 2014. 11858. Type strain: strain IGBL1 = CECT 8061 = Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 25436. 66: 4422-4432. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4422-4432. Acidipropionibacterium damnosum (Lucena-Padrós et al. 2014) Scholz and Acidipropionibacterium thoenii (van Niel Kilian 2016, comb. nov. 1928) Scholz and Kilian 2016, comb. nov. Basonym: Propionibacterium damnosum Basonym: Propionibacterium thoenii van Niel Lucena-Padrós et al. 2014. 1928. Type strain: strain IGBL13 = CECT 8062 = Type strain: strain ATCC 4874 = CCM 1865 = DSM 25450. CCUG 28149 = CGMCC 1.2228 = CIP Reference: Int. J. Syst. Evol. Microbiol., 2016, 103029 = DSM 20276 = JCM 6437 = LMG 66: 4422-4432. 16731 = NCIMB 5966. Reference: Int. J. Syst. Evol. Microbiol., 2016, Acidipropionibacterium jensenii (van Niel 66: 4422-4432. 1928) Scholz and Kilian 2016, comb. nov. Basonym: Propionibacterium jensenii van Niel Cutibacterium acnes (Gilchrist 1900) Scholz 1928. and Kilian 2016, comb. nov. Type strain: strain ATCC 4868 = CCUG 48883 Basonym: Propionibacterium acnes (Gilchrist = CGMCC 1.2229 = CIP 103028 = DSM 1900) Douglas and Gunter 1946. 20535 = NCIMB 8071. Type strain: strain ATCC 6919 = BCRC 10723 Reference: Int. J. Syst. Evol. Microbiol., 2016, = CCUG 1794 = CECT 5684 = CGMCC 66: 4422-4432. 1.5003 = CIP 53.117 = DSM 1897 = JCM 6425 = KCTC 3314 = LMG 16711 = NBRC Acidipropionibacterium microaerophilum 107605 = NCTC 737 = NRRL B-4224 = (Koussémon et al. 2001) Scholz and Kilian VKM Ac-1450. 2016, comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Propionibacterium microaerophilum 66: 4422-4432. Koussémon et al. 2001. Type strain: strain M5 = CIP 109962 = CNCM Cutibacterium avidum (Eggerth 1935) Scholz I-2360 = DSM 13435. and Kilian 2016, comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Propionibacterium avidum (Eggerth

S21 1935) Moore and Holdeman 1969. Type strain: strain ATCC 25577 = CCUG Glutamicibacter mysorens (Nand and Rao 36754 = CIP 103261 = DSM 4901 = NBRC 1972) Busse 2016, comb. nov. 15671 = NCIMB 702585 = NCTC 11864. Basonym: Arthrobacter mysorens Nand and Reference: Int. J. Syst. Evol. Microbiol., 2016, Rao 1972. 66: 4422-4432. Type strain: strain ATCC 33408 = CIP 102716 = DSM 12798 = JCM 11565 = KCTC 3381 Cutibacterium granulosum (Prévot 1938) = LMG 16219 = NBRC 103060 = NCIMB Scholz and Kilian 2016, comb. nov. 10583. Basonym: Propionibacterium granulosum Reference: Int. J. Syst. Evol. Microbiol., 2016, (Prévot 1938) Moore and Holdeman 1970. 66: 9-37. Type strain: strain ATCC 25564 = BCRC 17368 = CCUG 32987 = CIP 103262 = DSM 20700 Glutamicibacter nicotianae = JCM 6498 = LMG 16726 = NBRC 15672 (Giovannozzi-Sermanni 1959) Busse 2016, = NCIMB 702586 = NCTC 11865. comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Arthrobacter nicotianae 66: 4422-4432. Giovannozzi-Sermanni 1959. Type strain: strain ATCC 15236 = BCRC 11219 Glutamicibacter ardleyensis (Chen et al. 2005) = CCM 1648 = CCUG 23842 = CGMCC Busse 2016, comb. nov. 1.1895 = CIP 82.107 = DSM 20123 = JCM Basonym: Arthrobacter ardleyensis Chen et al. 1333 = KCTC 3382 = LMG 16305 = NBRC 2005. 14234 = NCIMB 9458. Type strain: strain An25 = CGMCC 1.3685 = Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 17432 = JCM 12921. 66: 9-37. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Glutamicibacter protophormiae (Lysenko 1959) Busse 2016, comb. nov. Glutamicibacter arilaitensis (Irlinger et al. Basonym: Brevibacterium protophormiae 2005) Busse 2016, comb. nov. Lysenko 1959; Arthrobacter protophormiae Basonym: Arthrobacter arilaitensis Irlinger et (Lysenko 1959) Stackebrandt et al. 1984. al. 2005. Type strain: strain ATCC 19271 = BCRC 12118 Type strain: strain Re117 = CIP 108037 = DSM = CCM 4749 = CGMCC 1.1921 = CIP 16368 = JCM 13566. 106987 = DSM 20168 = JCM 1973 = KCTC Reference: Int. J. Syst. Evol. Microbiol., 2016, 3385 = LMG 16324 = NBRC 12128 = 66: 9-37. NCIMB 12765 = VKM Ac-2104. Reference: Int. J. Syst. Evol. Microbiol., 2016, Glutamicibacter bergerei (Irlinger et al. 2005) 66: 9-37. Busse 2016, comb. nov. Basonym: Arthrobacter bergerei Irlinger et al. Glutamicibacter soli (Roh et al. 2008) Busse 2005. 2016, comb. nov. Type strain: strain Ca106 = CCUG 52342 = Basonym: Arthrobacter soli Roh et al. 2008. CIP 108036 = DSM 16367 = JCM 13567. Type strain: strain SYB2 = KCTC 19291 = Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 19449. 66: 9-37. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Glutamicibacter creatinolyticus (Hou et al. 1998) Busse 2016, comb. nov. Glutamicibacter uratoxydans (Stackebrandt et Basonym: Arthrobacter creatinolyticus Hou et al. 1984) Busse 2016, comb. nov. al. 1998. Basonym: Arthrobacter uratoxydans Type strain: strain CCM 4673 = CIP 105749 = Stackebrandt et al. 1984. DSM 15881 = JCM 10102 = KCTC 9903 = Type strain: strain ATCC 21749 = BCRC 14857 LMG 22054. = CGMCC 1.2821 = CIP 102367 = DSM Reference: Int. J. Syst. Evol. Microbiol., 2016, 20647 = JCM 11944 = KCTC 3482 = LMG 66: 9-37. 16220 = NBRC 15515 = NCIMB 702282 =

S22 VKM Ac-1979. Type strain: strain ATCC 14264 = CCM 4967 = Reference: Int. J. Syst. Evol. Microbiol., 2016, CCUG 23889 = CECT 4207 = CIP 107004 = 66: 9-37. DSM 20138 = JCM 12267 = LMG 3659 = LMG 7254 = NBRC 15514 = NCPPB 1228 Hoyosella subflava (Wang et al. 2010) Hamada = VKM Ac-1987. et al. 2016, comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Amycolicicoccus subflavus Wang et 66: 9-37. al. 2010. Type strain: strain DQS3-9A1 = CGMCC Paenarthrobacter nicotinovorans (Kodama et 4.3532 = DSM 45089 = JCM 17490 = al. 1992) Busse 2016, comb. nov. NBRC 109087. Basonym: Arthrobacter nicotinovorans Reference: Int. J. Syst. Evol. Microbiol., 2016, Kodama et al. 1992. 66: 4711-4715. Type strain: strain SAM 1563 = ATCC 49919 = CGMCC 1.1933 = CIP 106990 = DSM 420 Mycobacterium abscessus subsp. massiliense = JCM 3874 = KCTC 9902 = LMG 16253 = (Adékambi et al. 2006) Tortoli et al. 2016, NBRC 15511 = VKM Ac-1988. comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Mycobacterium massiliense 66: 9-37. Adékambi et al. 2006. Type strain: strain CCUG 48898 = CIP 108297 Paenarthrobacter nitroguajacolicus = DSM 45103 = KCTC 19086 = JCM (Kotoučková et al. 2004) Busse 2016, comb. 15300. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Arthrobacter nitroguajacolicus 66: 4471-4479. Kotoučková et al. 2004. Type strain: strain G2-1 = CCM 4924 = CIP Paenarthrobacter aurescens (Phillips 1953) 108435 = DSM 15232 = JCM 14115. Busse 2016, comb. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Arthrobacter aurescens Phillips 66: 9-37. 1953. Type strain: strain ATCC 13344 = BCRC 12110 Paenarthrobacter ureafaciens (Krebs and = CCM 1649 = CCUG 23839 = CCUG Eggleston 1939) Busse 2016, comb. nov. 23885 = CGMCC 1.1892 = CIP 102364 = Basonym: Arthrobacter ureafaciens (Krebs and DSM 20116 = JCM 1330 = KCTC 3378 = Eggleston 1939) Clark 1955. LMG 3815 = NBRC 12136 = NCIMB 8912 Type strain: strain ATCC 7562 = BCRC 10368 = NRRL B-2879 = VKM Ac-1105. = CCM 1644 = CGMCC 1.1897 = CIP 67.3 Reference: Int. J. Syst. Evol. Microbiol., 2016, = DSM 20126 = JCM 1337 = KCTC 3387 = 66: 9-37. LMG 3812 = NBRC 12140 = NCIMB 7811 = NCTC 7811 = VKM Ac-1121. Paenarthrobacter histidinolovorans (Adams Reference: Int. J. Syst. Evol. Microbiol., 2016, 1954) Busse 2016, comb. nov. 66: 9-37. Basonym: Arthrobacter histidinolovorans Adams 1954. Paeniglutamicibacter antarcticus (Pindi et al. Type strain: strain ATCC 11442 = BCRC 12111 2010) Busse 2016, comb. nov. = CCUG 23888 = CGMCC 1.1924 = CIP Basonym: Arthrobacter antarcticus Pindi et al. 106988 = DSM 20115 = JCM 2520 = KCTC 2010. 3380 = LMG 3822 = NBRC 15510 = Type strain: strain SPC26 = DSM 29880 = NCIMB 9541 = VKM Ac-1978. JCM 18952 = LMG 24542 = NCCB 100228. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. 66: 9-37.

Paenarthrobacter ilicis (Collins et al. 1982) Paeniglutamicibacter cryotolerans (Ganzert et Busse 2016, comb. nov. al. 2011) Busse 2016, comb. nov. Basonym: Arthrobacter ilicis Collins et al. Basonym: Arthrobacter cryotolerans Ganzert et 1982. al. 2011.

S23 Type strain: strain LI3 = DSM 22826 = JCM (Westerberg et al. 2000) Busse 2016, comb. 17806 = NCCB 100315. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Arthrobacter chlorophenolicus 66: 9-37. Westerberg et al. 2000. Type strain: strain A6 = ATCC 700700 = CIP Paeniglutamicibacter gangotriensis (Gupta et 107037 = DSM 12829 = JCM 12360 = al. 2004) Busse 2016, comb. nov. KCTC 9906 = NCIMB 13794. Basonym: Arthrobacter gangotriensis Gupta et Reference: Int. J. Syst. Evol. Microbiol., 2016, al. 2004. 66: 9-37. Type strain: strain Lz1y = CIP 108630 = DSM 15796 = JCM 12166. Pseudarthrobacter defluvii (Kim et al. 2008) Reference: Int. J. Syst. Evol. Microbiol., 2016, Busse 2016, comb. nov. 66: 9-37. Basonym: Arthrobacter defluvii Kim et al. 2008. Paeniglutamicibacter kerguelensis (Gupta et Type strain: strain 4C1-a = DSM 18782 = al. 2004) Busse 2016, comb. nov. KCTC 19209. Basonym: Arthrobacter kerguelensis Gupta et Reference: Int. J. Syst. Evol. Microbiol., 2016, al. 2004. 66: 9-37. Type strain: strain KGN15 = CIP 108629 = DSM 15797 = JCM 12165. Pseudarthrobacter equi (Yassin et al. 2011) Reference: Int. J. Syst. Evol. Microbiol., 2016, Busse 2016, comb. nov. 66: 9-37. Basonym: Arthrobacter equi Yassin et al. 2011. Type strain: strain IMMIB L-1606 = CCUG Paeniglutamicibacter psychrophenolicus 59597 = DSM 23395 = JCM 19107. (Margesin et al. 2004) Busse 2016, comb. Reference: Int. J. Syst. Evol. Microbiol., 2016, nov. 66: 9-37. Basonym: Arthrobacter psychrophenolicus Margesin et al. 2004. Pseudarthrobacter niigatensis (Ding et al. Type strain: strain AG31 = CIP 108593 = DSM 2009) Busse 2016, comb. nov. 15454 = JCM 13568 = LMG 21914. Basonym: Arthrobacter niigatensis Ding et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2009. 66: 9-37. Type strain: strain LC4 = CCTCC AB 206012 = DSM 28855 = JCM 30147. Paeniglutamicibacter sulfureus (Stackebrandt Reference: Int. J. Syst. Evol. Microbiol., 2016, et al. 1984) Busse 2016, comb. nov. 66: 9-37. Basonym: Arthrobacter sulfureus Stackebrandt et al. 1984. Pseudarthrobacter oxydans (Sguros 1954) Type strain: strain ATCC 19098 = CGMCC Busse 2016, comb. nov. 1.1898 = CIP 106986 = DSM 20167 = JCM Basonym: Arthrobacter oxydans Sguros 1954. 1338 = KCTC 3196 = LMG 16694 = NBRC Type strain: strain ATCC 14358 = BCRC 11573 12678 = NCIMB 10355 = NRRL B-14730. = CCUG 17757 = CECT 386 = CGMCC Reference: Int. J. Syst. Evol. Microbiol., 2016, 1.1925 = CIP 107005 = DSM 20119 = JCM 66: 9-37. 2521 = KCTC 3383 = LMG 3816 = NBRC 12138 = NCIMB 9333 = VKM Ac-1114. Parafrigoribacterium mesophilum (Dastager Reference: Int. J. Syst. Evol. Microbiol., 2016, et al. 2008) Kong et al. 2016, comb. nov. 66: 9-37. Basonym: Frigoribacterium mesophilum Dastager et al. 2008. Pseudarthrobacter phenanthrenivorans Type strain: strain MSL-08 = DSM 19442 = (Kallimanis et al. 2009) Busse 2016, comb. JCM 19547 = KCTC 19311. nov. Reference: Int. J. Syst. Evol. Microbiol., 2016, Basonym: Arthrobacter phenanthrenivorans 66: 5252-5259. Kallimanis et al. 2009. Type strain: strain Sphe3 = DSM 18606 = JCM Pseudarthrobacter chlorophenolicus 16027 = LMG 23796.

S24 Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 13068 = JCM 11943 = KCTC 9908. 66: 9-37. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Pseudarthrobacter polychromogenes (Schippers-Lammertse et al. 1963) Busse Pseudoglutamicibacter cumminsii (Funke et al. 2016, comb. nov. 1997) Busse 2016, comb. nov. Basonym: Arthrobacter polychromogenes Basonym: Arthrobacter cumminsii Funke et al. Schippers-Lammertse et al. 1963. 1998. Type strain: strain ATCC 15216 = BCRC 12114 Type strain: strain DMMZ 445 = ATCC 700218 = CCUG 23891 = CGMCC 1.1927 = CIP = CCM 4574 = CCUG 36788 = CIP 104907 106989 = DSM 20136 = JCM 2523 = KCTC = DSM 10493 = JCM 11675 = KCTC 9904. 3384 = LMG 16679 = LMG 3821 = NBRC Reference: Int. J. Syst. Evol. Microbiol., 2016, 15512 = NCIMB 10267 = VKM Ac-1955. 66: 9-37. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Pseudopropionibacterium propionicum (Buchanan and Pine 1962) Scholz and Kilian Pseudarthrobacter scleromae (Huang et al. 2016, comb. nov. 2005) Busse 2016, comb. nov. Basonym: Propionibacterium propionicum Basonym: Arthrobacter scleromae Huang et al. (Buchanan and Pine 1962) Charfreitag et al. 2005. 1988; Arachnia propionica (Buchanan and Type strain: strain YH-2001 = CGMCC 1.3601 Pine 1962) Pine and Georg 1969. = CIP 108992 = DSM 17756 = JCM 12642. Type strain: strain ATCC 14157 = CCUG 4939 Reference: Int. J. Syst. Evol. Microbiol., 2016, = CIP 101941 = DSM 43307 = JCM 5830 = 66: 9-37. LMG 19873 = NBRC 14587 = NCTC 12967 = VKM Ac-1449. Pseudarthrobacter siccitolerans Reference: Int. J. Syst. Evol. Microbiol., 2016, (SantaCruz-Calvo et al. 2013) Busse 2016, 66: 4422-4432. comb. nov. Basonym: Arthrobacter siccitolerans Sphaerimonospora mesophila (Nonomura and SantaCruz-Calvo et al. 2013. Ohara 1971) Mingma et al. 2016, comb. nov. Type strain: strain 4J27 = CECT 8257 = DSM Basonym: Microbispora mesophila (Nonomura 28024 = LMG 27359. and Ohara 1971) Zhang et al. 1998; Reference: Int. J. Syst. Evol. Microbiol., 2016, Thermomonospora mesophila Nonomura and 66: 9-37. Ohara 1971. Type strain: strain T-1 = ATCC 27303 = BCRC Pseudarthrobacter sulfonivorans (Borodina et 12464 = CIP 105593 = DSM 43048 = JCM al. 2002) Busse 2016, comb. nov. 3151 = KCTC 9241 = NBRC 14179 = Basonym: Arthrobacter sulfonivorans Borodina NCIMB 11544 = NRRL B-16986 = VKM et al. 2002. Ac-1953. Type strain: strain ALL = ATCC BAA-112 = Reference: Int. J. Syst. Evol. Microbiol., 2016, DSM 14002 = JCM 13520. 66: 1735-1744. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 9-37. Sphaerimonospora thailandensis (Duangmal et al. 2014) Mingma et al. 2016, comb. nov. Pseudoglutamicibacter albus (Wauters et al. Basonym: Microbispora thailandensis 2000) Busse 2016, comb. nov. Duangmal et al. 2014. Basonym: Arthrobacter albus Wauters et al. Type strain: strain NN276 = BCC 41490 = 2000. NBRC 107569 = NRRL B-24806. Type strain: strain CF43 = ATCC BAA-273 = Reference: Int. J. Syst. Evol. Microbiol., 2016, CCM 4905 = CCUG 43812 = CIP 106791 = 66: 1735-1744.

EMENDATION OF CLASS

S25 Rubrobacteria Suzuki 2013 emend. Foesel et Thermoleophilia Suzuki and Whitman 2013 al. 2016 emend. Foesel et al. 2016 Type order: Rubrobacterales Rainey et al. Type order: Thermoleophilales Reddy and 1997. Garcia-Pichel 2009. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 652-665. 66: 652-665. A member of the phylum Actinobacteria. A member of the phylum Actinobacteria.

EMENDATION OF ORDER

Gaiellales Albuquerque et al. 2012 emend. Solirubrobacterales Reddy and Garcia-Pichel Foesel et al. 2016 2009 emend. Foesel et al. 2016 Type genus: Gaiella Albuquerque et al. 2012. Type genus: Solirubrobacter Singleton et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2003. 66: 652-665. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the class Rubrobacteria. 66: 652-665. A member of the class Thermoleophilia. Rubrobacterales Rainey et al. 1997 emend. Reddy and Garcia-Pichel 2009 emend. Zhi et Thermoleophilales Reddy and Garcia-Pichel al. 2009 emend. Foesel et al. 2016 2009 emend. Foesel et al. 2016 Type genus: Rubrobacter Suzuki et al. 1989. Type genus: Thermoleophilum Zarilla and Reference: Int. J. Syst. Evol. Microbiol., 2016, Perry 1986. 66: 652-665. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the class Rubrobacteria. 66: 652-665. A member of the class Thermoleophilia.

EMENDATION OF FAMILY

Conexibacteraceae Stackebrandt 2005 emend. Stackebrandt 2004 emend. Zhi et al. 2009 Zhi et al. 2009 emend. Foesel et al. 2016 emend. Foesel et al. 2016 Type genus: Conexibacter Monciardini et al. Type genus: Rubrobacter Suzuki et al. 1989. 2003. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 652-665. 66: 652-665. A member of the order Rubrobacterales. A member of the order Solirubrobacterales. Solirubrobacteraceae Stackebrandt 2005 Gaiellaceae Albuquerque et al. 2012 emend. emend. Zhi et al. 2009 emend. Foesel et al. Foesel et al. 2016 2016 Type genus: Gaiella Albuquerque et al. 2012. Type genus: Solirubrobacter Singleton et al. Reference: Int. J. Syst. Evol. Microbiol., 2016, 2003. 66: 652-665. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the order Gaiellales. 66: 652-665. A member of the order Solirubrobacterales. Patulibacteraceae Takahashi et al. 2006 emend. Zhi et al. 2009 emend. Foesel et al. 2016 Thermoleophilaceae Stackebrandt 2005 emend. Type genus: Patulibacter Takahashi et al. 2006. Zhi et al. 2009 emend. Foesel et al. 2016 Reference: Int. J. Syst. Evol. Microbiol., 2016, Type genus: Thermoleophilum Zarilla and 66: 652-665. Perry 1986. A member of the order Solirubrobacterales. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 652-665. Rubrobacteraceae Rainey et al. 1997 emend. A member of the order Thermoleophilales.

S26 EMENDATION OF GENUS

Actinomadura Lechevalier and Lechevalier 1968 emend. Zhao et al. 2016 Hoyosella Jurado et al. 2009 emend. Hamada Type species: Actinomadura madurae (Vincent et al. 2016 1894) Lechevalier and Lechevalier 1968. Type species: Hoyosella altamirensis Jurado et Reference: Antonie van Leeuwenhoek, 2015, al. 2009. 108: 1331-1339; List of changes in Reference: Int. J. Syst. Evol. Microbiol., 2016, taxonomic opinion no. 24 [Int. J. Syst. Evol. 66: 4711-4715. Microbiol., 2016, 66: 2469-2470]. A member of the family Mycobacteriaceae. A member of the family Thermomonosporaceae. Hoyosella Jurado et al. 2009 emend. Li et al. 2016 Arthrobacter Conn and Dimmick 1947 emend. Type species: Hoyosella altamirensis Jurado et Koch et al. 1995 emend. Busse 2016 al. 2009. Type species: Arthrobacter globiformis (Conn Reference: Int. J. Syst. Evol. Microbiol., 2016, 1928) Conn and Dimmick 1947. 66: 4716-4722. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the family Mycobacteriaceae. 66: 9-37. A member of the family Micrococcaceae. Microbacterium Orla-Jensen 1919 emend. Takeuchi and Hatano 1998 emend. Blastococcus Ahrens and Moll 1970 emend. Krishnamurthi et al. 2012 emend. Alves et al. Urzì et al. 2004 emend. Lee 2006 emend. 2015 emend. Fidalgo et al. 2016 Hezbri et al. 2016 Type species: Microbacterium lacticum Type species: Blastococcus aggregatus Ahrens Orla-Jensen 1919. and Moll 1970. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4492-4500. 66: 4864-4872. A member of the family Microbacteriaceae. A member of the family Geodermatophilaceae. Ornithinicoccus Groth et al. 1999 emend. Demequina Yi et al. 2007 emend. Ue et al. Zhang et al. 2016 2011 emend. Park et al. 2016 Type species: Ornithinicoccus hortensis Groth Type species: Demequina aestuarii Yi et al. et al. 1999. 2007. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 1894-1899. 66: 4197-4203. A member of the family Intrasporangiaceae. A member of the family Demequinaceae. Propionibacterium Orla-Jensen 1909 emend. Flexivirga Anzai et al. 2012 emend. Kang et al. Charfreitag et al. 1988. emend. Scholz and 2016 Kilian 2016 Type species: Flexivirga alba Anzai et al. 2012. Type species: Propionibacterium freudenreichii Reference: Int. J. Syst. Evol. Microbiol., 2016, van Niel 1928. 66: 3594-3599. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the family Dermacoccaceae. 66: 4422-4432. A member of the family Propionibacteriaceae. Hamadaea Ara et al. 2008 emend. Chu et al. 2016 Salininema Nikou et al. 2015 emend. Li et al. Type species: Hamadaea tsunoensis (Asano et 2016 al. 1989) Ara et al. 2008. Type species: Salininema proteolyticum Nikou Reference: Int. J. Syst. Evol. Microbiol., 2016, et al. 2015. 66: 1818-1822. Reference: Int. J. Syst. Evol. Microbiol., 2016, A member of the family Micromonosporaceae. 66: 2558-2565.

S27 A member of the family Glycomycetaceae. al. 2015. Reference: Int. J. Syst. Evol. Microbiol., 2016, Tenggerimyces Sun et al. 2015 emend. Li et al. 66: 1499-1505. 2016 A member of the family Nocardioidaceae. Type species: Tenggerimyces mesophilus Sun et

EMENDATION OF SPECIES

Arthrobacter roseus Reddy et al. 2002 emend. Corynebacterium mastitidis Busse 2016 Fernandez-Garayzabal et al. 1997 emend. Type strain: strain CMS 90r = CIP 107726 = Bernard et al. 2016 DSM 14508 = JCM 11881 = MTCC 3712 = Type strain: strain S-8 = CCUG 38654 = CECT NCIMB 14039. 4843 = CIP 105509 = DSM 44356 = LMG Reference: Int. J. Syst. Evol. Microbiol., 2016, 19040 = NBRC 16160. 66: 9-37. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 2803-2812. Blastococcus aggregatus Ahrens and Moll 1970 emend. Urzì et al. 2004 emend. Hezbri Frankia alni (Woronin 1866) Von Tubeuf 1895 et al. 2016 emend. Nouioui et al. 2016 Type strain: strain ATCC 25902 = DSM 4725 = Type strain: strain ACN14a = CECT 9034 = JCM 12602 = NBRC 107747 = NCIMB DSM 45986. 1849. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 5201-5210. 66: 4864-4872. Hoyosella altamirensis Jurado et al. 2009 Blastococcus endophyticus Zhu et al. 2013 emend. Hamada et al. 2016 emend. Hezbri et al. 2016 Type strain: strain OFN S31 = CIP 109864 = Type strain: strain YIM 68236 = CCTCC AA DSM 45258 = JCM 18112 = NBRC 109631. 209045 = DSM 45413 = JCM 17896 = Reference: Int. J. Syst. Evol. Microbiol., 2016, KCTC 19998. 66: 4711-4715. Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4864-4872. Mycobacterium abscessus (Moore and Frerichs 1953) Kusunoki and Ezaki 1992 emend. Blastococcus jejuensis Lee 2006 emend. Leao et al. 2011 emend. Tortoli et al. 2016 Hezbri et al. 2016 Type strain: strain ATCC 19977 = CCUG Type strain: strain KST3-10 = DSM 19597 = 20993 = CCUG 27982 = CIP 104536 = DSM JCM 15614 = KCCM 42251 = NRRL 44196 = JCM 13569 = NCTC 13031 = TMC B-24440. 1543. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4864-4872. 66: 4471-4479.

Blastococcus saxobsidens Urzì et al. 2004 Salininema proteolyticum Nikou et al. 2015 emend. Hezbri et al. 2016 emend. Li et al. 2016 Type strain: strain BC444 = DSM 44509 = Type strain: strain Miq-4 = IBRC-M 10908 = JCM 13239 = NRRL B-24246. LMG 28391. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4864-4872. 66: 2558-2565.

EMENDATION OF SUBSPECIES

S28 Mycobacterium abscessus subsp. abscessus (Moore and Frerichs 1953) Leao et al. 2011 Mycobacterium abscessus subsp. bolletii emend. Tortoli et al. 2016 (Adékambi et al. 2006) Leao et al. 2011 Type strain: strain ATCC 19977 = CCUG emend. Tortoli et al. 2016 20993 = CCUG 27982 = CIP 104536 = DSM Type strain: strain BD = CCUG 50184 = CIP 44196 = JCM 13569 = NCTC 13031 = TMC 108541 = DSM 45149 = JCM 15297. 1543. Reference: Int. J. Syst. Evol. Microbiol., 2016, Reference: Int. J. Syst. Evol. Microbiol., 2016, 66: 4471-4479. 66: 4471-4479.

SYNONYM

Brevibacterium massiliense Roux and Raoult Reference: Int. J. Syst. Evol. Microbiol., 2016, 2009 pro synon. Brevibacterium 66: 5519-5522. ravenspurgense Mages et al. 2009 Reference: Int. J. Syst. Evol. Microbiol., 2016, Paraglycomyces xinjiangensis Luo et al. 2015 66: 4440-4444. pro synon. Salininema proteolyticum Nikou et al. 2015 Oceanitalea nanhaiensis Fu et al. 2012 pro Reference: Int. J. Syst. Evol. Microbiol., 2016, synon. Georgenia satyanarayanai Srinivas et 66: 2558-2565. al. 2012

NEOTYPE STRAIN

Actinobaculum massiliense Greub and Raoult DSM 100580. 2006 Reference: Int. J. Syst. Evol. Microbiol., 2016, Neotype strain: strain FC3 = CSUR P1982 = 66: 2702-2703.

S29 Hamada award 2016

Exploiting the potential of biosynthesis of natural products by actinomycetes: bacterial interaction-driven natural product discovery and biosynthetic machinery

Shumpei Asamizu

Graduate School of Agricultural and Life Sciences, The University of Tokyo

1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

Actinomycetes are a major source of natural bioactive to human health that include global expansion of multi-drug products with important chemical and biological properties. resistance (Martens and Demain, 2017) and neglected Recent genome analysis has revealed the previously tropical diseases in developing countries (Buscaglia et al., unrecognized huge potential of biosynthesis of natural 2015). New strategies and technologies are becoming products by actinomycetes. It is now generally accepted that indispensable for the effective discovery and/or generation of more microbial chemical and biosynthetic diversities novel bioactive compounds (Katz and Baltz, 2016). remain undiscovered. Increased knowledge of microbial After the publication of genome sequences for the model production of bioactive compounds would increase the actinomycetes Streptomyces coelicolor A3(2) (Bentley et al., repertoire of useful agents. Moreover, bioengineering 2002) and the avermectin-producer S. avermitilis MA-4680 involving genes and enzymes would generate new useful (Ikeda et al., 2003) in the early 2000s, we quickly recognized compounds. However, this potential remains challenging. that actinomycetes possess more potential to produce secondary Methods have been developed to activate biosynthetic gene metabolites than previously thought (Nett et al., 2009). The clusters that are normally silent or poorly expressed under database maintained by the National Center for Biotechnology laboratory conditions. Section I highlights research aimed Information (NCBI) now contains several hundred at the discovery of novel compounds by using co-culture, actinomycete genome sequences (including draft genome especially the interaction between intergeneric sequences). Scrutiny of these sequences using bioinformatics actinobacteria. Additionally, we discuss the importance of tools like antiSMASH and PRISM has readily revealed putative understanding the natural enzymatic assembly of complex secondary metabolite gene clusters (Blin et al., 2017; Skinnider small molecules in order to exploit new resources for et al., 2017). Twenty to forty putative secondary metabolite biocatalysis, genes, and chemistry, which can lead to the biosynthetic gene clusters have been identified in the genomes creation of new antibiotics. This knowledge could enable the of individual strains. Most remain uncharacterized. The rational design of metabolic pathways to produce “artificial” accepted view is that their remains vast chemical and natural products in engineered bacteria. Section II details biosynthetic diversities in the microbial world. current research on the biosynthetic mechanisms of C7N Understanding and exploiting the uncharacterized aminocyclitol natural products having a unique chemical microbial chemistry would drive the discovery of new chemical structure and important biological activities. agents to control bioactivity. Furthermore, the use of bioengineering tools including those directed at genes and Section I enzymes would allow the creation of new useful compounds Bacterial interaction-driven (Katz and Baltz, 2016). However, these goals remain natural product discovery challenged by the difficulty activating the relevant gene clusters and identifying their products. Methodologies to activate Actinomycetes are an important source of natural products biosynthetic gene clusters that are silent or poorly expressed in with significant chemical and biological properties. Bioactive laboratory conditions have been developed (Ochi, 2017). This natural products isolated from actinomycetes have been used review will focus on research that aims to achieve effective widely and include antibacterial, antifungal, and antiparasitic discovery of novel compounds by using a co-culture strategy, agents for treatment of infectious diseases; insecticides and especially using an interaction of intergeneric actinobacteria herbicides for agricultural purposes; and anticancer and involving Streptomyces species and mycolic acid-containing immunosuppressive drugs for clinical chemotherapy (Demain bacteria. and Sanchez, 2009). However, the discovery rate of new antibiotics has been declining in recent decades, despite these Bacterial co-culture as a means of discovering natural successes and the more contemporary emerging/rising threats products

S30 Isolated actinomycetes are traditionally cultured alone as a was implicated, given the similar effects on production of mono-culture to search for new natural products. However, the pigments in liquid culture by Corynebacterineae, which also natural environment where actinobacteria live involve complex possess mycolic acids (Onaka et al., 2011). To test the idea that interactions at the intra- and inter-species, -genetic, and - contact with the mycolic acid-containing cell membrane was kingdom levels (van der Meij et al., 2017). Yet, little is known necessary to induce production of undecylprodigiosin, dead about how the specialized metabolites encoded by cryptic gene cells of T. pulmonis, which were intact and still contained clusters is used for the actinomycete life cycle in the complex, mycolic acids, were prepared by formaldehyde fixation and real-world environment (Traxler and Kolter, 2015). Discovery gamma-irradiation (Asamizu et al., 2015). The dead cells did of useful bioactive natural products based on mono-culture has not induce the pigment formation by S. lividans, suggesting the been successful, although this strategy is laborious. With the involvement of another factor (Asamizu et al., 2015). (Fig. 1) increasing evidence that bacterial interaction can drive the When co-cultures of S. lividans and T. pulmonis or activation of previously quiescent secondary metabolite gene Rhodococcus opacus B4 were closely observed by scanning clusters (Bertrand et al., 2014), development and understanding electron microscopy, co-aggregation was evident (Asamizu et of the induction of specialized metabolites during co-culture has al., 2015). (Fig. 1) The presence of an intimate relationship become recognized as a research priority. between microbes to alter the specialized pattern of metabolites has been described in the co-culture of the fungi Aspergillus Interaction between Streptomyces lividans and mycolic nidulans or A. fumigatus with Streptomyces rapamycinicus acid-containing bacteria (Netzker et al., 2015). Although contact-mediated interaction in Using the pigment production by Streptomyces lividans microbes has not been well characterized yet (Stubbendieck and TK23 as indication of specialized metabolites activation, Onaka Straight, 2016; Westhoff et al., 2017), close distance et al. (2011) discovered Tsukamurella pulmonis TP-B0596 from recognition may well be beneficial in ecosystems such as soil. a laboratory bacterial culture collection. T. pulmonis phylogenetically belongs to the order Actinomycetales, the Physicochemical-based discovery of specialized metabolites same order as Streptomyces species. T. pulmonis from combined-culture phylogenetically diverges to the suborder Corynebacterineae. Comparison of high performance liquid chromatography Most species in this suborder possess specific long chain fatty (HPLC) patterns between culture extracts from mono-cultures acids, mycolic acids, on the cell outer membrane (Jackson, and combined-cultures has shown that T. pulmonis can 2014). markedly change its production of secondary metabolites. Examination of the interaction between S. Examination of 112 strains of actinomycetes isolated from soil lividans and T. pulmonis in a dual culture agar plate experiment samples collected in the Hokuriku district of Japan revealed revealed a response by S. lividans featuring production of the new metabolite peaks in 41 strains, with increased production red pigmented compound undecylprodigiosin upon contact of metabolites in 61 strains. In total, 99 strains showed variation with T. pulmonis colonies (Asamizu et al., 2015; Onaka et al., in the HPLC traces (Onaka et al., 2011). The same study 2011). (Fig. 1) The production of the red pigment required the documented that some of the soil-isolated actinomycetes physical contact between the strains, since when the strains showed the induced antibiotic activity in combined-culture. were physically separated during liquid culture using a dialysis Among them, the antibiotic alchivemycin A was isolated from membrane, the red pigment was not produced (Onaka et al., a co-culture of Streptomyces sp. S522 (NBRC109436) and T. 2011). The mycolic acids on the T. pulmonis outer membrane pulmonis (Igarashi et al., 2010; Onaka et al., 2011). (Fig. 2)

Figure 1. Interaction between S. lividans TK23 and T. pulmonis TP-B0596 or R. opacus B4. Growing colony of T. pulmonis or R. opacus induced production of red pigments by S. lividans upon contact (A). R. opacus was observed to adhere on the mycelium of S. lividans during the liquid culture (B).

S31 More recently, HPLC trace comparison-based screening of et al., 2015). Subtraction of a self-organizing heat map revealed the new compounds from combined-cultures enabled the differentially expressed metabolites; using several co-culture identification of new metabolites using Streptomyces species challengers, the authors found the mycolic acid-containing isolated from soil or obtained from culture collection that were bacterial strain, Rhodococcus wratislaviensis, induced co-cultured with T. pulmonis TP-B0596. These include Nocardiopsis sp. FU40 ΔapoS strain to produce cytotoxic indolocarbazole arcyriaflavin E production by S. cinnamoneus ciromicin A and B (Derewacz et al., 2015). (Fig. 2) NBRC13823 (Hoshino et al., 2015c), cytotoxic butanolides Traxler et al. (2013) used imaging mass spectrometry to chojalactone A–C from Streptomyces sp. CJ-5 (Hoshino et al., visualize the secreted metabolome of S. coelicolor A3(2) and 2015b), and macrolactams niizalactam A–C from Streptomyces Amycolatopsis sp. AA4. They found that in consequence of sp. NZ-6 (Hoshino et al., 2015a). (Fig. 2) amychelin production by Amycolatopsis sp. AA4, S. coelicolor Similar co-culturing methods were reported by Bachmann A3(2) react to produce several new acyl-desferrioxamines, and co-workers, in which comparative metabolomics enabled which are different from regular siderophores found to produce visualization of differentially expressed metabolites produced by S. coelicolor A3(2). The study highlight competition of by S. coelicolor A3(2) with several known secondary bacteria using siderophores for Fe uptake (Traxler et al., 2013). metabolites inducing factors, such as rare earth elements, (Fig. 2) streptomycin/rifampicin resistance, and co-cultures (Goodwin

Figure 2. Structure of induced specialized metabolites found in combined-culture, and other co-culture between intergeneric actinobacteria. Undecylprodigiosins and actinorhodins from S. lividans and T. pulmonis, alchivemycin A from Streptomyces sp. S522 and T. pulmonis, 5aTHQs and streptoaminals from Streptomyces sp. HEK616 and T. pulmonis, arcyliaflavin E from S. cinnamoneus NRBC13823 and T. pulmonis, Cyojalactone A-C from Streptomyces sp. CJ-5 and T. pulmonis, Niizalactame A-C from Streptomyces sp. NZ-6 and T. pulmonis, Ciromicin A and B from Nocardiopsis sp. FU40ΔapoS and R. wratislaviensis (study by Derewacz DK, et al. 2015), acyl-desferrioxamines from S. coelicolor A3(2) and Amycolatopsis sp. AA4 (study by Traxler MF, et al. 2013)

S32 Combined-culture with S. lividans harboring exogenous competition for survival between the two bacteria. However, gene cluster knowledge is limited and more studies are needed to address a As production of several endogenous secondary number of questions. What are the stimuli? How do bacteria metabolites from S. lividans TK23 (RED and ACT) were sense the stimuli? How do the stimuli lead to the production of effectively induced by T. pulmonis, effects for production of specialized metabolites? Are the interactions observed in exogenous gene cluster coding metabolites were examined laboratory co-culture relevant to real-world ecosystems? (Onaka et al., 2015). Interestingly, when S. lividans mutant strains harboring exogenous gene clusters were cultured with T. Section II: pulmonis, production of the exogenous secondary metabolites C7N aminocyclitol natural products goadsporin (Onaka et al., 2001), staurosporine (Onaka et al.,

2002), and rebeccamycin (Onaka et al., 2003) were The C7N aminocyclitol family of natural products has significantly increased in mixed cultures compared to mono- clinically important biological activities; therefore, C7N culture (Onaka et al., 2015). The method was applied for gene aminocyclitol natural products and their derivatives have been disruptants; significantly improved accumulation of goadsporin used in agricultural and pharmaceutical fields (Mahmud, 2003). C (a glutamylated-Ser4 variant of goadsporin B) was observed The antifungal agent validamycin A (Iwasa et al., 1970) and α- (Ozaki et al., 2016). This improved production of shunt glucosidase inhibitor acarbose (Schmidt et al., 1977) are intermediates contributed to the elucidation of important prominent examples of C7N aminocyclitols, and these bacterial biosynthetic steps in the thiopeptide family of ribosomally secondary metabolites are associated with pseudo- synthesized peptide natural products (Ozaki et al., 2016). oligosaccharides (or simply pseudosugars), which function as sugar hydrolase inhibitors (Gloster and Davies, 2010; Mahmud,

Bioactivity-guided discovery of natural products from 2003). One unique structural feature in this family is their C7N combined-cultures carbasugar scaffold, primarily valienamine moieties. In Sugiyama et al. (2015) searched for the yeast membrane addition to validamycin A and acarbose, typical compounds that interacting small molecules from combined-culture induced contain valienamine moieties also include the trehalase bacterial metabolites. The extracts from combined-cultures of inhibitor salbostatin (Vertesy et al., 1994), α-amylase inhibitor actinomycetes isolated from Hegura Island, Ishikawa, Japan, trestatins (Yokose et al., 1983), and antibiotic pyralomicin and T. pulmonis were tested against wild-type fission yeast and (Kawamura et al., 1995) (Fig. 3). Along with the recent ergosterol premature mutants. This bioactivity-guided discovery of novel cyclitol natural products and an screening successfully led to the isolation of eight 5-alkyl- understanding of their origins, biosynthesis, biological 1,2,3,4-tetrahydroquinolines (5aTHQs) with diversity in the activities, and ecological functions, the structurally more alkyl side chains (Sugiyama et al., 2015). (Fig. 2) 5aTHQ-7n diverse family of C7N aminocyclitols, which includes the was shown to be the most potent antifungal agent of the eight cytotoxic carbasugar cetoniacytone A (Schlorke et al., 2002), congeners. Moreover, 5aTHQ-9i showed selective antifungal antibiotic epoxyquinomicin (Tsuchida et al., 1996), and activity to the wild-type, but not against ergosterol premature kirkamide (Pinto-Carbo et al., 2016; Sieber et al., 2015), has mutants (Sugiyama et al., 2015). The results suggested that been identified. Mycosporine-like amino acids (e.g., shinorine) 5aTHQs bioactivity may involve targeting of the yeast cell are natural sunscreen compounds that have the same precursor membrane. Sugiyama et al. (2016) also isolated broad-spectrum and share homologous biosynthetic enzymes in the initial step antibiotic streptoaminals from the combined-culture extracts (Asamizu et al., 2012; Balskus and Walsh, 2010; Mahmud, containing a similar alkyl chain pattern to that of 5aTHQs 2003; Miyamoto et al., 2014; Wu et al., 2007) (Fig. 3). In this (Sugiyama et al., 2016). (Fig. 2) Production of streptoaminals review, we discuss the recently investigated biosynthetic steps was enhanced by combined-culture. The structural similarity of C7N aminocyclitol natural products. In particular, we discuss between 5aTHQs and streptoaminals implies that both the pseudoglycosyltranferase-catalyzed C-N bond formation compounds share their biosynthetic routes. Interestingly, process during validamycin A biosynthesis and the catalytic 5aTHQs were only detected in the combined-culture of divergence of sugar phosphate cyclases leading to the

Streptomyces sp. HEK616 and T. pulmonis. However, 5aTHQs generation of various C7N cyclitol natural products. did not show antibacterial activities. Further biosynthesis studies may provide insight into the molecular mechanism of Biosynthesis of the antifungal trehalase inhibitor the specific production of 5aTHQs by Streptomyces sp. validamycin A HEK616 during co-culture with T. pulmonis. Validamycin A was originally isolated from Streptomyces hygroscopicus subsp. limoneus by a group from Takeda Future perspectives Pharmaceutical Co. in the early 1970s (Iwasa et al., 1970). The We have observed a variety of specialized metabolites compound inhibits the growth of the fungus Rhizoctonia solani, induced during co-culture. However, the link between the which causes sheath blight disease in rice (Iwasa et al., 1970) induced small molecules and the function within the co-cultured by inhibiting the activity of trehalase (Asano et al., 1987). bacteria remains unclear. One bacterium can cause significant Therefore, the antifungal agent validamycin A has been used as changes in the culture (living) environment during the growth a crop protectant in East/Southeast Asia. Later, the α- process, which can incidentally trigger the production of glucosidase inhibitor voglibose (Fig. 3) was synthesized from irrelative compounds. Interestingly, some of the induced validamycin A and used to treat type-II insulin-independent compounds have antibiotic activity, which could reflect the diabetes.

S33 Figure 3. Structure of C7N aminocyclitol natural products.

The biosynthetic gene cluster for validamycin A was first phosphate. cloned from Streptomyces hygroscopicus subsp. jingangensis To produce GDP-valienol, the first ketoreduction of 5008 (val cluster) (Yu et al., 2005). The first step in the valienone 7-phosphate yields valienol 7-phosphate through the secondary metabolism of validamycin is catalyzed by 2-epi-5- function of ValN, a putative bifunctional oxidoreductase (Fig. epi-valiolone synthase (EEVS), which converts D- 4A). Next, phosphomutation of valienol 7-phosphate to give sedoheptulose 7-phosphate (SH7P), a pentose phosphate valienol 1-phosphate is thought to occur through the activity of pathway intermediate, to 2-epi-5-epi-valiolone (EEV; Fig. 4A). ValO, a putative bifunctional phosphomutase/phosphatase. The first EEVS (AcbC) was found and characterized in the Then, using valienol 1-phosphate and GTP, the nucleotidylation acarbose biosynthetic pathway from Actinoplanes sp. SE reaction produces GDP-valienol through catalysis by ValB in 50/110 by precursor feeding studies (Mahmud et al., 1999) and vitro (Yang et al., 2011). biochemical studies (Stratmann et al., 1999). Later, ValA was To produce validamine 7-phosphate, transamination of found to be EEVS in the validamycin A biosynthetic pathway valienone 7-phosphate yields valienamine 7-phosphate through (Yu et al., 2005). the activity of ValM, a putative pyridoxal 5′-phosphate (PLP)-

Interestingly, the biosynthesis of C7N aminocyclitols was dependent aminotransferase (Fig. 4A). Then, reduction of suggested to diverge into several assembly lines, including valienamine 7-phosphate to give validamine 7-phosphate those for validamycin A, acarbose (Rockser and Wehmeier, occurs through catalysis by ValN (Xu et al., 2009a). The 2009), salbostatin (Choi et al., 2008), pyralomicin 1a (Flatt et coupling reaction of the two cyclitol units (GDP-valienol and al., 2013), and cetoniacytone A (Wu et al., 2009), after the validamine 7-phosphate) will be described in the next section. formation of EEV. In this review, the validamycin A After formation of validoxylamine A, ValG catalyzes the O- biosynthetic pathway will be described in detail. A summary of glucosyltransferase reaction to produce the final product the biosynthetic pathways for other cyclitols can be found in validamycin A (Bai et al., 2006; Xu et al., 2008). previous reviews (Flatt and Mahmud, 2007; Mahmud, 2009). Several oxygenated validamycin derivatives have also The second step in validamycin A biosynthesis, been isolated from cultures of validamycin A-producing epimerization of EEV to generate 5-epi-valiolone, was found to Streptomyces species (Mahmud, 2003). VldW, an α- be catalyzed by cyclitol epimerase ValD in vitro (Xu et al., ketoglutarate and Fe(II) dependent dioxygenase from 2009b) (Fig. 4A). Next, dehydration of 5-epi-valiolone to Streptomyces hygroscopicus var. linoneus (vld cluster) (Singh produce valienone is thought to be catalyzed by ValK, a putative et al., 2006), was characterized and found to catalyze the dehydratase (Cui et al., 2016). Then, ATP-dependent production of validamycin B from validamycin A by the regio- phosphorylation of valienone to produce valienone 7-phosphate /stereo-selective oxygenation of the methylene carbon is catalyzed by cyclitol kinase ValC in vitro (Minagawa et al., (Almabruk et al., 2012) (Fig. 4A). 2007). After the formation of valienone 7-phosphate, the pathway was predicted to branch into two pathways to generate Nonglycosidic C-N bond formation in validamycin A two different C7 cyclitol units, GDP-valienol and validamine 7- biosynthesis is catalyzed by pseudoglycosyltransferase

S34 Figure 4. Proposed biosynthetic pathway of validamycin A from Streptomyces hygroscopicus (A), and proposed reaction mechanism of pseudoglycosyltransferase, VldE (B).

ValL/VldE shares 29% identity (41% similarity) with transition state”, it remains uncertain whether this prediction is trehalose 6-phosphate synthase (OtsA) from Streptomyces true. coelicolor A3(2). OtsA is a retaining-type glycosyltransferase VldB (cyclitol nucleotidyltransferase) (Yang et al., 2011), that synthesizes trehalose 6-phosphate with an α,α-1,1′- VldE (trehalose 6-phosphate synthase homolog), and VldH glycosidic bond using nucleotide diphosphate (NDP)-glucose (putative phosphatase) from Streptomyces hygroscopicus subsp. and glucose 6-phosphate, and OtsB dephosphorylates trehalose limoneus were expressed in Escherichia coli and purified as 6-phosphate to give trehalose (Giaever et al., 1988). Yang et al. recombinant proteins to test the hypothesis (Asamizu et al., showed that ValB catalyzes the formation of GDP-valienol from 2011). First, VldB was confirmed to be a nucleotidyltransferase valienol 1-phosphate and GTP (Yang et al., 2011). Hence, that gave GDP-valienol from valienol 1-phosphate and GTP researchers hypothesized that validoxylamine A 7′-phosphate (Asamizu et al., 2011) (Fig 4A). Next, to examine the catalytic (mimic of trehalose 6-phosphate) may be produced by the activity of VldE, the enzyme was incubated with the possible coupling of GDP-valienol (mimic of NDP-glucose) and substrates validamine 7-phosphate and GDP-valienol. High- validamine 7-phosphate (mimic of glucose 6-phosphate; Fig. performance liquid chromatography (HPLC) and mass 4A). However, for the retaining-type glycosyltransferase spectrometry (MS) analyses showed that VldE catalyzed the reaction, such as in OtsA, an internal return (SNi)-like reaction formation of validoxylamine 7′-phosphate with net retention of mechanism in which the donor sugar molecule is in the an anomeric-like configuration by accepting GDP-valienol and oxocarbenium transition state, may exist during the reaction validamine 7-phosphate as substrates (Asamizu et al., 2011) (Errey et al., 2010; Lee et al., 2011). Since the cyclitol (Fig. 4B). Interestingly, VldE did not accept GDP-glucose and molecules (valienol moiety) cannot form the “oxocarbenium glucose 6-phosphate as substrates to produce trehalose 6-

S35 Figure 5. Catalytic divergence in sugar phosphate cyclase family enzymes. SH7P cyclases (EEVS, EVS, and DDGS) involved in biosynthesis

of C7N aminocyclitol natural products share homologies with DHQS, aDHQS, and DOIS. These enzymes represent the family of sugar phosphate cyclase involved in primary and secondary metabolism. (Abbreviation: DAHP; 3-Deoxy-D-arabinoheptulosonate 7-phosphate, DHQ; 3- dehydroquinic acid, aminoDAHP; 3,4-dideoxy-4-amino-D-arabinoheptulosonate 7-phosphate, 3,5-AHBA; 3-amino-5-hydroxybenzoic acid, SH7P; D-sedoheptulose 7-phosphate, EEV; 2-epi-5-epi-valiolone, 2EV; 2-epi-valiolone, DDG; desmethyl-4-deoxygadusol, MAA; mycosporine- like amino acid, G6P; glucose 6-phosphate, DOI; 2-deoxy-scyllo-inosose.) phosphate. This indicated the narrow substrate tolerance of the similar to the cocrystallized X-ray structure of dedicated VldE enzyme in validamycin A biosynthesis glycosyltransferase OtsA from E. coli with UDP and the (Abuelizz and Mahmud, 2015; Asamizu et al., 2011). Upon the mechanistic inhibitor validoxylamine 7′-phosphate (Cavalier et addition of VldH, formation of validoxylamine A by al., 2012; Errey et al., 2010; Lee et al., 2011). These structural consumption of validoxylamine A 7′-phosphate was observed comparisons indicated that OtsA, the true retaining-type by HPLC and MS analysis (Asamizu et al., 2011). These glycosyltransferase OtsA, and the pseudoglycosyltransferase biochemical investigations could clearly demonstrate the VldE exhibited similar reaction mechanisms. Thus, analogous interesting enzymatic conversion steps in validamycin A to the proposed reaction mechanism for the retaining-type biosynthesis (Asamizu et al., 2011). glycosyltransferase OtsA (Lee et al., 2011), hydrogen bonding To investigate the reaction mechanism through which interactions among the donor phosphate group and acceptor VldE catalyzes the coupling of the “nonsugar” donor molecule nucleophile were proposed to enable front side attack to (GDP-valienol) and the acceptor molecule (validamine 7- promote the substitution reaction while retaining its phosphate) with the retention of stereochemistry, a series of configuration, and the double bond π-electron of the donor VldE crystal structures cocrystallizing with different ligands nonsugar was predicted to mimic the transition state in the were solved (Cavalier et al., 2012). The overall X-ray crystal PsGT reaction (Asamizu et al., 2011; Cavalier et al., 2012) (Fig. structure of VldE showed a typical GT-B fold with two β/α/β 4B). Rossmann-like folding domains (Lairson et al., 2008). The Based on biochemical and structural studies, Abuelizz and products of VldE, i.e., GDP and validoxylamine A 7′-phosphate, Mahmud produced domain-swapped chimeric proteins between were found to bind in the cleft formed by the two domains, VldE and OtsA from Streptomyces coelicolor A3(2) and indicating the position of the active center. Interestingly, the examined the catalytic activity of the “chimeras” to elucidate cocrystallized structure of VldE with GDP and validoxylamine their substrate tolerances (Abuelizz and Mahmud, 2015). By A 7′-phosphate showed a ligand-binding conformation that was swapping the substrates, they showed the potential for

S36 biocatalysis of engineered proteins and demonstrated the liquid chromatography-high-resolution MS (LC-HRMS) importance of the amine group as a better nucleophile to (Asamizu et al., 2013). The identified specific metabolite with promote the coupling reaction (Abuelizz and Mahmud, 2015). m/z 314 was purified, and the chemical structure was Further characterization of other “PsGT” candidates found determined to be validoxylamine A (Asamizu et al., 2013) (Fig. in acarbose, salbostatin, pyralomicins, and many other 4A). Thus, there are pathways with different steric courses in compounds in genome databases will expand our knowledge of the assembly pathway for C7N aminocyclitol natural products. the unique PsGT-catalyzed reaction. Since true Other sugar phosphate cyclase (SPC) members are also glycosyltransferase enzymes are ubiquitous in both primary and involved in the biosynthesis of mycosporine-like amino acids secondary metabolism (Elshahawi et al., 2015), protein by several cyanobacteria (Wu et al., 2007). Balskus and Walsh engineering of a glycosyltransferase to be a PsGT catalyst identified a biosynthetic gene cluster for the mycosporine-like would allow the creation of useful tools to generate novel amino acid shinorine in cyanobacteria (Balskus and Walsh, pseudoglycosylated conjugants. 2010) (Fig. 5). They demonstrated the enzyme activities of Ava_3858 (desmethyl-4-deoxygadusol synthase [DDGS]) and

A divergent pathway for production of C7N cyclitols from Ava_3857 (S-adenosylmethionine [SAM]-dependent sedoheptulose 7-phosphate methyltransferase) from the cyanobacteria Anabaena variabilis During genome mining to search for structurally novel ATCC 29413, which generated 4-deoxygadusol from SH7P aminocyclitol natural products, PsGT-containing gene clusters (Balskus and Walsh, 2010) (Fig. 5). DDGS and EEVS share with 3-dehydroquinate synthase (DHQS) homolog genes were homology with each other (Wu et al., 2007); therefore, unexpectedly found in the genomes of several bacteria researchers tested whether there was a common intermediate (Asamizu et al., 2012). Previously characterized gene clusters during the DDGS reaction, in which additional dehydration was for C7N aminocyclitol natural products were found to all involved. The reaction of Ava_3858 and Npun_5600 (DDGSs contain EEVS genes (Bai et al., 2006; Choi et al., 2008; Flatt from the cyanobacteria Nostoc punctiforme PCC 73102) and Mahmud, 2007; Stratmann et al., 1999; Wu et al., 2007; Wu (Balskus and Walsh, 2010) was traced by in situ NMR, and the et al., 2009; Yu et al., 2005). Although EEVS genes share 1H NMR signals showed only the chemical shifts for DDG, homology with 3-dehydroquinate synthase (Stratmann et al., indicating that no detectable intermediate was generated during 1999; Wu et al., 2007), the identified putative proteins (e.g., the entire reaction (Asamizu et al., 2012). Amir_2000 from the actinomycete Actinosynnema mirum and To gain insights into how three homologous enzymes Staur_3140 from the myxobacteria Stigmatella aurantiaca (EEVS, EVS, and DDGS) catalyze different cyclization DW4/3-1) showed more similarity in their fingerprint amino reactions using the same substrate SH7P, the crystal structures acid residues to DHQS than to EEVS (Kean et al., 2014; Wu et of ValA (EEVS: 4P53) (Kean et al., 2014) and Ava_3858 al., 2007) and existed in different phylogenetic clades from (DDGS: 5TPR) (Osborn et al., 2017a) were solved. The crystal known EEVSs (Asamizu et al., 2012; Osborn et al., 2015). structures of ValA and Ava_3858 were found to be cocrystalized To characterize the catalytic function of the genes, with NAD+ and Zn2+ and showed folds that were similar to those Amir_2000 and Staur_3140 were examined for enzymatic of DHQS (Carpenter et al., 1998). A comparison of the amino activities using purified recombinant proteins expressed in E. acid residues forming the catalytic pocket among EEVS, DDGS, coli. First, as annotated in the NCBI database, the proteins were and DHQS provided some insights into the fingerprint amino tested for their DHQS activity by incubating them with 3- acid residues used for accurate annotation of gene function for deoxy-D-arabinoheptulosonate 7-phosphate (DAHP), a similar enzymes (Kean et al., 2014; Osborn et al., 2017a). substrate of DHQS in the shikimate pathway; however, no However, swapping the amino acid residues that are specifically consumption of DAHP was observed (Asamizu et al., 2012). conserved in each enzyme (L267E/D281A/H360T for ValA; Then, SH7P was tested as a substrate and incubated with the E254L/A268D/T347H for Ava_3858) did not convert the proteins. Consumption of SH7P was observed; however, activity of the enzyme; thus, it remained unclear how the surprisingly, the converted products showed different chemical additional dehydration process could proceed in the DDGS properties from EEV, the most likely product to be generated reaction (Osborn et al., 2017a).

(Asamizu et al., 2012). Comparative analysis with synthetic C7 In addition, during genome mining using the EEVS cyclitols which exhibited different stereochemistries, by gas sequence as a probe, unexpectedly, homologous genes were chromatography (GC)/MS and in situ nuclear magnetic found in the genome of vertebrates, such as fish, birds, reptiles, resonance (NMR) revealed that the true product was 2-epi- and amphibians (Osborn et al., 2015). Interestingly, the putative valiolone (2EV), a diastereomer of EEV (Asamizu et al., 2012) EEVS genes from animals were flanked by a putative protein (Fig. 5). Interestingly, in situ NMR gave a two sets of 1H NMR with an oxidoreductase (Ox) domain and a methyltransferase signals for the products, which were confirmed by a quantum (MT) domain, and were also flanked by putative transcriptional mechanics/molecular mechanics (QM/MM) study to be derived regulators (Osborn et al., 2015). The genes for the EEVS from two stable conformations of 2EV (Asamizu et al., 2012). homolog and the Ox-MT di-domain protein were synthesized To further elucidate the metabolite(s) of the gene cluster that based on the zebrafish (Danio rerio) sequences and expressed encodes the 2EV synthase (EVS) gene (amir_2000) and the in E. coli. The purified recombinant DrEEVS was shown to PsGT homolog gene (amir_1997), both genes in A. mirum were synthesize EEV from SH7P. Furthermore, co-incubation with disrupted individually, and the metabolites from culture of the Ox-MT and SAM resulted in the formation of gadusol from wild-type gene, amir_1997 disruptant, and amir_2000 EEV (Osborn et al., 2015) (Fig. 5). Gadusol is a natural disruptant were analyzed by comparative metabolomics using sunscreen compound that possesses UV-resistance activity

S37 (Shick and Dunlap, 2002). Gadusol was originally identified in pseudoglycosyltransferase VldE. Chem. Biol. 22, 724-733. fish eggs and was believed to accumulate from consumption in Almabruk, K.H., et al. (2012). The alpha-ketoglutarate/Fe(II)- the diet (Shick and Dunlap, 2002). However, this study dependent dioxygenase VldW is responsible for the provided insights into the animal de novo synthetic pathway of formation of validamycin B. Chembiochem. 13, 2209-2211. a natural C7 cyclitol sunscreen compound that exists not only in Asamizu, S., et al. (2011). Pseudoglycosyltransferase catalyzes prokaryotes but also in higher organisms, such as vertebrates nonglycosidic C-N coupling in validamycin A biosynthesis. (Osborn et al., 2015). J. Am. Chem. Soc. 133, 12124-12135. Until recently, the EEVS involved in the biosynthesis of Asamizu, S., et al. (2012). Evolutionary divergence of

C7N aminocyclitol natural products from actinomycetes was the sedoheptulose 7-phosphate cyclases leads to several distinct only characterized SH7P cyclase, a family of enzymes that cyclic products. J. Am. Chem. Soc. 134, 12219-12229. convert SH7P to carbocyclic molecules, such as EEV, 2EV, and Asamizu, S., et al. (2013). Comparative metabolomic analysis DDG (Osborn et al., 2017b). However, genomic and of an alternative biosynthetic pathway to pseudosugars in biochemical investigations have revealed that SH7P cyclase is Actinosynnema mirum DSM 43827. Chembiochem. 14, distributed in a wide range of species, including actinomycetes, 1548-1551. cyanobacteria (Asamizu et al., 2012; Balskus and Walsh, 2010), Asamizu, S., et al. (2015). Killing of Mycolic Acid-Containing myxobacteria (Asamizu et al., 2012), and vertebrates, such as Bacteria Aborted Induction of Antibiotic Production by zebrafish (D. rerio) (Osborn et al., 2015). Further Streptomyces in Combined-Culture. PLoS One. 10, bioinformatics analysis of homologous genes for SH7P cyclase e0142372. revealed that this gene is widely distributed in a variety of Asano, N., et al. (1987). Effect of validamycins on organisms (Osborn et al., 2017a; Osborn et al., 2017b). These glycohydrolases of Rhizoctonia solani. J. Antibiot. 40, 526- recently investigated SH7P cyclase genes will provide a 532. template for easier access to gene clusters for new C7N Bai, L., et al. (2006). Functional analysis of the validamycin aminocyclitols that are buried inside the growing genome biosynthetic gene cluster and engineered production of databases. validoxylamine A. Chem. Biol. 13, 387-397. Balskus, E.P., and Walsh, C.T. (2010). The genetic and Conclusion and perspective molecular basis for sunscreen biosynthesis in cyanobacteria. In this section, I reviewed recent progress in research on Science. 329, 1653-1656. the biosynthesis of C7N aminocyclitol validamycin A. These Bentley, S.D., et al. (2002). Complete genome sequence of the recent investigations have revealed the intriguing assembly model actinomycete Streptomyces coelicolor A3(2). Nature. pathways of these secondary metabolites by unique enzymes. 417, 141-147. The recent expansion of genome databases has been a driving Bertrand, S., et al. (2014). Metabolite induction via force in the discovery of unprecedented biological, chemical, microorganism co-culture: a potential way to enhance and catalytic repertoires, which can provide challenges to create chemical diversity for drug discovery. Biotechnol Adv. 32, novel bioactive “artificial” natural products. Further 1180-1204. accumulation of knowledge is indispensable and will facilitate Blin, K., et al. (2017). antiSMASH 4.0-improvements in the development of new technologies to achieve the aim of chemistry prediction and gene cluster boundary creating novel cell factories that can synthesize complex small identification. Nucleic Acids Res. molecules. Buscaglia, C.A., et al. (2015). Neglected Tropical Diseases in the Post-Genomic Era. Trends Genet. 31, 539-555. Acknowledgments Carpenter, E.P., et al. (1998). Structure of dehydroquinate I am grateful to Prof. Hiroyasu Onaka at The University of synthase reveals an active site capable of multistep catalysis. Tokyo, and Prof. Taifo Mahmud at Oregon State University for Nature. 394, 299-302. their kind support. I would like to acknowledge Prof. Andrew Cavalier, M.C., et al. (2012). Mechanistic insights into Karplus at Oregon State University, Prof. Yong-Hwan Lee at validoxylamine A 7'-phosphate synthesis by VldE using the Louisiana State University, Dr. Hideaki Kakeya at Kyoto structure of the entire product complex. PLoS One. 7, e44934. University, Dr. Satoh Katsuya at QST, and Dr. Kanae Teramoto Choi, W.S., et al. (2008). Genetic organization of the putative at JOEL Ltd. for research collaboration. I would like to thank salbostatin biosynthetic gene cluster including the 2-epi-5- Dr. Taro Ozaki, Dr. Shohei Hayashi, and Dr. Yoshinori Sugai epi-valiolone synthase gene in Streptomyces albus ATCC for their kind support. I would also like to thank all present and 21838. Appl. Microbiol. Biotechnol. 80, 637-645. past laboratory members at The University of Tokyo and Cui, L., et al. (2016). De Novo biosynthesis of beta- Oregon State University for their kind support. Finally, I would valienamine in engineered Streptomyces hygroscopicus 5008. like to thank the Society of Actinomycetes Japan for the ACS Synth. Biol. 5, 15-20. Hamada Award. Demain, A.L., and Sanchez, S. (2009). Microbial drug discovery: 80 years of progress. J. Antibiot. 62, 5-16. Derewacz, D.K., et al. (2015). Mapping Microbial Response References Metabolomes for Induced Natural Product Discovery. ACS Chem. Biol. 10, 1998-2006. Abuelizz, H.A., and Mahmud, T. (2015). Distinct substrate Elshahawi, S.I., et al. (2015). A comprehensive review of specificity and catalytic activity of the glycosylated bacterial natural products. Chem. Soc. Rev. 44,

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S40 Publication of Award Lecture

The Society for Actinomycetes Japan Hamada Award 2016,

Dr. Shumpei Asamizu

“Exploiting the potential of biosynthesis of natural products by actinomycetes: bacterial interaction-driven natural product discovery and biosynthetic machinery” Actinomycetologica (2017) 31 [1], S30-S40.

Graduate School of Agricultural and Life Sciences, The University of Tokyo 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan

S41 Hamada award 2016

Enzyme involved in the biosynthesis of a unique polyketide in actinomycetes

Takashi Kawasaki

College of Pharmaceutical Sciences, Ritsumeikan University,

1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan

INTRODUCTION

Angucycline antibiotics are a large group of naturally occurring aromatic polyketides of microbial origin (Rohr, J et al., 1992; Krohn, K et al., 1997). They exhibit a wide range of biological activities including antibacterial, antiviral, antitumor, and enzyme inhibitory activities. Although they contain a benz[a]anthraquinone skeleton, the structural diversity of these antibiotics is very broad with a wide variety of oxidation states. Hatomarubigins A, B, C, and D (Fig. 1) belong to the angucycline family and reverse colchicine resistance in multidrug-resistant tumor cells (Hayakawa, Y et al., 1991). Among them, hatomarubigin D is a unique hatomarubigin C dimer with a methylene linkage. This dimer has not been reported previously and little is known about the mechanism of methylene bridge formation between the two aromatic rings. In this review, I describe studies that identified a gene cluster for hatomarubigin biosynthesis in Streptomyces sp. 2238-SVT4, conversion of new metabolite hatomarubigin E by hrbU encoding an O-methyltransferase, and hrbY genes involved in the biosynthesis of hatomarubigin D in a unique dimeric angucycline.

Cloning of a gene cluster for angucycline biosynthesis from Streptomyces sp. 2238-SVT4 Angucycline biosynthetic gene clusters commonly contain both aromatase and cyclase genes, which construct the tetracyclic angucycline frameworks. The primers for PCR amplification were designed from conserved amino acid sequences of the aromatase (lanL/jadD/urdL) and cyclase (lanF/jadI/urdF) genes in the landomycin biosynthetic gene cluster from S. cyanogenus S136 (Westrich, L., et al. 1999), jadomycin biosynthetic gene cluster from S. venezuelae ISP5230 (Han, L., K., et al. 1994; Chen, Y. H., et al. 2005), and urdamycin biosynthetic gene cluster from S. fradiae Tü2717 (Decker, H., et al. 1995; Faust, B., D., et al. 2000). The aromatase and cyclase gene fragments were amplified by PCR with Streptomyces sp. 2238-SVT4 genomic DNA. These gene fragments shared amino acid identities of 79% with Fig. 1. Structures of angucycline antibiotics aromatase UrdL and 76% with cyclase JadI. Two cosmid clones were selected from a cosmid library of Streptomyces sp. 2238-SVT4 by colony hybridization and Southern blot

S42 Fig. 2. Hatomarubigin biosynthesis gene cluster from Streptomyces sp. 2238-SVT4. Arrow shows DNA regions used for heterologous expression. analysis using the aromatase and cyclase gene fragments as Expression of part of the hrb gene cluster in S. lividans probes. These cosmids were sequenced to identify a 35-kbp To establish the function of hrbs, the expression plasmid DNA region consisting of 33 open reading frames (ORFs) as pWHM-HR containing hrbR1 to hrbX was constructed and shown in Fig. 2. Seventeen ORFs were homologous to introduced into S. lividans TK23. S. lividans harboring angucycline biosynthesis genes previously reported by pWHM-HR or an empty vector pWHM3 was cultivated, and homology searching (Table 1). Based on their positions and the mycelial extract was analyzed by high-performance liquid deduced functions, 30 ORFs were designated as hrb genes and chromatography (HPLC). S. lividans expressing hrb genes consisted of a gene cluster for angucycline biosynthesis in produced hatomarubigins A, B, and C and rubiginone B2 as Streptomyces sp. 2238-SVT4. The hrb cluster contained the show in Fig. 3. However, a peak for hatomarubigin D was not ketosynthase, chain length determinant factor, acyl carrier detected in HPLC. These results demonstrated that the hrb protein, ketoreductase, aromatase, cyclase, genes consist of a gene cluster for hatomarubigin biosynthesis O-methyltransferase, oxidoreductase, and oxygenase genes. in Streptomyces sp. 2238-SVT4. Three regulatory genes, hrbR1, hrbR2, and hrbR3, and a transporter gene, hrbT, were found in the cluster (Kawasaki, T., Estimated function of hrb genes in hatomarubigin et al. 2010a). biosynthesis hrbI resembles lanV, a 6-ketoreductase gene involved in

TABLE 1. Deduced functions of the hrb gene products. ORF Size (aa) Homologous protein (angucycline biosynthesis gene product) Identity (%) Similarity (%) Proposed function

1 160 Alanine rich protein of Streptomyces ambofaciens 42 49 R1 71 Regulatory protein of Streptomyces coelicolor 42 50 Regulator A 281 Unknown protein in an angucycline biosynthesis gene cluster(Aur1O) 42 53 B 112 Cyclase (JadI) 70 79 Cyclase C 423 Ketosynthase (JadA) 82 87 Minimal PKS D 107 Predicted protein of Coprinopsis cinerea 42 66 E 404 Chain length determinant factor (JadB) 71 79 Minimal PKS F 234 Methyltransferase of Caulobacter crescentus 34 44 G 680 Oxygenase-reductase (LndM2) 57 66 6-Hydroxylase H 373 Oxygenase of Rhodococcus sp. 45 60 Oxygenase I 262 Reductase (LanV) 49 62 6-Ketoreductase J 226 Oxygenase of Pseudomonas fluorescens 35 59 Oxygenase K 208 Oxygenase (JadG) 30 48 Oxygenase L 358 Oxygenase (LndZ5) 42 56 Oxygenase M 87 Acyl carrier protein (JadC) 72 80 Minimal PKS N 302 Type II thioesterase of Mycobacterium liflandii 35 53 O 261 Ketoreductase (JadD) 79 88 10-Ketoreductase P 311 Aromatase (UrdL) 79 87 Aromatase Q 279 Phosphopantetheinyl transferase (JadM) 53 63 R 460 Oxygenase (LanE) 64 75 Oxygenase S 254 Ketoacyl reductase of Frankia alni 44 55 Oxidoreductase T 434 Transporter (UrdJ2) 47 55 Transporter U 343 O-Methyltransferase of Streptomyces glaucescens 38 55 O-Methyltransferase V 263 Short-chain dehydrogenase / reductase of Thermobifida fusca 43 57 Oxidoreductase W 193 Reductase (LanO) 58 65 11-Hydroxylase X 377 Oxygenase (LanZ5) 36 49 11-Hydroxylase R2 200 Transcriptional regulator of Saccharopolyspora erythraea 46 62 Regulator Y 365 Oxidoreductase of Saccharopolyspora erythraea 47 60 Methylene bridge formation R3 240 Repressor-response regulator (JadR1) 61 72 Regulator Z1 304 N5,N10-Methylenetetrahydromethanopterin reductase of Rhodococcus sp. 32 48 Oxidoreductase Z2 334 Aldo/keto reductase of Actinosynnema mirum 71 81 Oxidoreductase 2 53 S-Adenosylmethionine synthase of Streptomyces clavuligerus 72 81 3 336 Carbohydrate kinase of Streptomyces rishiriensis 55 63 aa, number of amino acids. PKS, polyketide synthase

S43 landomycin biosynthesis (Mayer, A., et al. 2005), and shows the same function. Hatomarubigins possess a methoxy group at the 8-position. hrbU is a candidate O-methyltransferase gene based on its homology to tcmO, an O-methyltransferase gene involved in tetracenomycin biosynthesis (Summers, R. G., et al. 1993). hrbG encodes an enzyme with putative oxygenase and reductase domains. Although its homologous gene urdM is involved in 12b-oxygenation in urdamycin biosynthesis (Faust, B. D., et al. 2000), C-12b bears no oxygen atom in hatomarubigins. A gene homologous to urdM, lndM2, is responsible for 6-hydroxylation in landomycin biosynthesis (Zhu, L., et al. 2005), and the oxygenase domain of HrbG displays the highest identity (57%) to that of LndM2, suggesting that HrbG catalyzes 6-hydroxylation of rubiginone

B2 to yield hatomarubigin A (Fig. 4). In landomycin biosynthesis, the two tandem genes lanZ4 and lanZ5 encoding oxidoreductase and oxygenase are involved in 11-hydroxylation (Ostash, B., et al. 2004). A gene homologous to lanZ5, hrbX, is followed by the reductase gene hrbW. These two gene products may catalyze 11-hydroxylation of rubiginone B2 to give hatomarubigin B (Fig. 4). The hrb cluster contains three regulatory gene candidates. HrbR1, HrbR2, and HrbR3 show sequence similarity with a putative regulatory protein of Streptomyces coelicolor (Bentley, S.D., et al. 2004), MarR-family regulator of Fig. 3. HPLC analysis of hatomarubigins produced by S. lividans expressing a part of the hatomarubigin biosynthesis gene cluster. Saccharopolyspora erythraea (Oliynyk, M., et al. 2007), and 1: rubiginone B2. 2: hatomarubigin B. 3: hatomarubigin A. atypical response regulator (JadR1) of Streptomyces 4: hatomarubigin C. venezuelae (Wang, L., et al. 2009), respectively.

Fig. 4. Proposed pathway for hatomarubigin biosynthesis.

S44 New metabolite hatomarubigin E, a biosynthetic intermediate of hatomarubigin C A new metabolite in the 2-day culture of Streptomyces sp. 2238-SVT4 detected by thin-layer chromatography and HPLC analyses. The molecular formula of hatomarubigin E was established as C19H16O5 by high-resolution fast-atom bombardment-mass spectrometry. The 13C- and 1H-NMR data of hatomarubigin E resembled those of hatomarubigin C, except for a methoxy group in hatomarubigin C (Fig. 1). We isolated the new metabolite hatomarubigin E, 8-demethyl hatomarubigin C (Kawasaki, T., et al. 2010b). In the hrb cluster, hrbU shows homology to an O-methyltransferase gene involved in tetracenomycin C biosynthesis. This indicates that hrbU catalyzes the methylation of hatomarubigin E. To confirm the function of hrbU, the recombinant enzyme was expressed in Escherichia coli. HrbU converted hatomarubigin E to hatomarubigin C, using S-(5′-adenosyl)-L-methionine as a cofactor (Fig. 5). This reaction corresponds to the 8-O-methylation step in hatomarubigin biosynthesis. HrbU contains a conserved motif for S-(5′-adenosyl)-L-methionine binding (DVGGARG) (Ingrosso, D., et al. 1989; Haydock,S.F., Fig. 5. HPLC analysis of hatomarubigins. et al. 1991; Madduri, K., et al. 1993). HrbU was found to (a): Mycelial acetone extract of 2-day cultured Streptomyces sp. 2238-SVT4. convert hatomarubigin E to hatomarubigin C during (b): standard sample of hatomarubigin C. hatomarubigin biosynthesis. (c): reaction mixture for hatomarubigin E conversion without HrbU. (d): reaction mixture of hatomarubigin E conversion with HrbU.

Conversion of hatomarubigin C to hatomarubigin D by HrbY Genes remaining in the cluster are candidates for methylene bridge formation in the production of hatomarubigin D. The gene cluster for hatomarubigin biosynthesis includes the oxidoreductase gene hrbY, which is present in hrbR3, a gene homologous to the regulator of angucycline biosynthesis (Table 1). HrbY was expressed in E. coli, and the purified recombinant HrbY was assayed for its ability to convert hatomarubigin C. Hatomarubigin C was converted into hatomarubigin D by HrbY, using methylcobalamin and NADPH as cofactors (Fig. 6) (Kawasaki, T., et al. 2010a). This reaction is the final step of Fig. 6. HPLC analysis of the conversion of hatomarubigin C into hatomarubigin D. hatomarubigin biosynthesis (Fig. 4). HrbY exhibited (A): hatomarubigin D. (B): reaction mixture without enzyme. homology to an FAD-dependent pyridine nucleotide-disulfide (C): reaction mixture with HrbY oxidoreductase of Saccharopolyspora erythraea (Oliynyk, M., methylene linkage. In the beginning of the first step, we et al. 2007) and contained conserved FAD and NAD(P)H cloned the gene cluster for angucycline biosynthesis from binding motifs (GGGYGGAAVAKALEAEADVILIDPRD Streptomyces sp. 2238-SVT4, a hatomarubigin producer. To and VLILGAGPVGLE; underlining indicates conserved identify the gene cluster involved in hatomarubigin amino acids) (Dym, O., et al. 2001). Because the reaction biosynthesis, a gene cluster of 25 genes (hrbR1-hrbX) was required NAD(P)H but not FAD, the recombinant enzyme expressed in S. lividans, and transformants produced may be purified as a complex with FAD. HrbY used hatomarubigin A, B, and C. Thus, we obtained the gene cluster methylcobalamin as a C donor for methylene bridge 1 involved in hatomarubigin biosynthesis. To further understand formation. Methylcobalamin is known to participate in several the biosynthesis of hatomarubigin, we isolated new metabolite enzymatic methyl group transfer reactions (Banerjee, R., et al. hatomarubigin E, 8-demethyl hatomarubigin C, following 2003). However, there is no similarity between HrbY and such 2-day culture of Streptomyces sp. 2238-SVT4. hrbU shows enzymes. These results indicate that HrbY is a novel enzyme homology to an O-methyltransferase gene. Therefore, HrbU that catalyzes methylene bridge formation between two likely catalyzes methylation of hatomarubigin. Indeed, angucycline molecules. recombinant HrbU converted hatomarubigin C to hatomarubigin E. Hatomarubigin C was converted to CONCLUSION hatomarubigin D of unique structure by HrbY, which used

methylcobalamin as a C1 donor for methylene bridge We attempted to elucidate the biosynthetic mechanism of formation. However, there is no similarity between HrbY and hatomarubigin D, which has a unique dimeric structure with a other enzymes that use methylcobalamin as a C1 donor. The

S45 enzymatic hatomarubigin C conversion reported here will D-aspartyl/Lisoaspartyl protein methyltransferase from facilitate future studies of the exact mechanism of methylene human erythrocytes. Common sequence motifs for bridge formation. protein, DNA, RNA and small molecule S-adenosylmethionine dependent methyltransferase. J. ACKNOWLEDGMENTS Biol. Chem. 264, 20131–20139. Kawasaki, T., et al. (2010a). Cloning and Characterization of I am very pleased to receive the prestigious Hamada a Gene Cluster for Hatomarubigin Biosynthesis in Award of SAJ (Society of Actinomycetes, Japan). This study Streptomyces sp. Strain 2238-SVT4. Appl. Environ. was mainly conducted at the Faculty of Pharmaceutical Microbiol. 76, 4201-4206. Sciences, Tokyo University of Science. I would like to express Kawasaki, T., et al. (2010b). Hatomarubigin E, a biosynthetic my gratitude to the members of the laboratory. I would like to intermediate of hatomarubigins C and a substrate of express my deepest gratitude and appreciation to Prof. Yoichi HrbU O-methyltransferase. The Journal of Antibiotics. 63, Hayakawa for providing suggestions and guidance. I am 725-727. deeply indebted to Prof. Tohru Dairi from Hokkaido Krohn, K., et al. (1997). Angucyclines: Total syntheses, new University for providing suggestions. I would like to thank structures, and biosynthetic studies of an emerging new everyone at SAJ. class of antibiotics. Top. Curr. Chem. 188, 127–195. Madduri, K., et al. (1993). Cloning and sequencing of a gene REFERNCES encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia

Banerjee, R., et al. (2003). The many faces of vitamin B12: coli. J. Bacteriol. 175, 3900–3904. catalysis by cobalamin-dependent enzymes. Annu. Rev. Mayer, A., et al. (2005). LanV, a bifunctional enzyme: Biochem. 72, 209-247. aromatase and ketoreductase during landomycin A Bentley, S. D., et al. (2004). SCP1, a 356,023 bp linear biosynthesis. Chembiochem. 6, 2312-2315. plasmid adapted to the ecology and developmental Oliynyk, M., et al. (2007). Complete genome sequence of the biology of its host, Streptomyces coelicolor A3(2). Mol. erythromycin-producing bacterium Saccharopolyspora Microbiol. 51, 1615-1628. erythraea NRRL23338. 2007. Nat. Biotechnol. 25, Chen, Y. H., et al. (2005). Functional analyses of oxygenases 447-453. in jadomycin biosynthesis and identification of JadH as a Ostash, B., et al. (2004). Generation of new landomycins by bifunctional oxygenase/dehydrase. J. Biol Chem. 280, combinatorial biosynthetic manipulation of the lndGT4 22508-22514. gene of the landomycin E cluster in S. globisporus. Chem. Decker, H., et al. (1995). Cloning and characterization of a Biol. 11, 547-555. polyketide synthase gene from Streptomyces fradiae Rohr, J., et al. (1992). Angucycline group antibiotics. Nat. Tü2717, which carries the genes for biosynthesis of the Prod. Rep. 9, 103–137. angucycline antibiotic urdamycin A and a gene probably Summers, R. G., et al. (1993). The tcmVI region of the involved in its oxygenation. J. Bacteriol. 177, 6126-6136. tetracenomycin C biosynthetic gene cluster of Dym, O., et al. (2001). Sequence-structure analysis of Streptomyces glaucescens encodes the tetracenomycin F1 FAD-containing proteins. Protein Sci. 10, 1712-1728. monooxygenase, tetracenomycin F2 cyclase, and, most Faust, B. D., et al. (2000). Two new tailoring enzymes, a likely, a second cyclase. J. Bacteriol. 175, 7571-7580. glycosyltransferase and an oxygenase, involved in Wang, L., et al. (2009). Autoregulation of antibiotic biosynthesis of the angucycline antibiotic urdamycin A in biosynthesis by binding of the end product to an atypical Streptomyces fradiae Tü2717. Microbiology 146, response regulator. Proc Natl Acad Sci USA. 106, 147-154. 8617-8622. Han, L. K., et al. (1994). Cloning and characterization of Westrich, L., et al. (1999). Cloning and characterization of a polyketide synthase genes for jadomycin B biosynthesis gene cluster from Streptomyces cyanogenus S136 in Streptomyces venezuelae ISP5230. Microbiology. 140, probably involved in landomycin biosynthesis. FEMS 3379-3389. Microbiol Lett. 170, 381-387. Hayakawa, Y., et al. (1991). Studies on the isotetracenone Zhu, L., et al. (2005). Identification of the function of gene antibiotics. IV. Hatomarubigins A, B, C and D, new lndM2 encoding a bifunctional oxygenase-reductase isotetracenone antibiotics effective against involved in the biosynthesis of the antitumor antibiotic multidrug-resistant tumor cells. J. Antibiot. 44, landomycin E by Streptomyces globisporus 1912 1179-1186. supports the originally assigned structure for Haydock, S. F., et al. (1991). Cloning and sequence analysis landomycinone. J. Org. Chem. 70, 631-638. of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferase. Mol. Gen. Genet. 230, 120–128. Ingrosso, D., et al. (1989). Sequence of the

S46 Publication of Award Lecture

The Society for Actinomycetes Japan Hamada Award 2016,

Dr. Takashi Kawasaki

“Enzyme involved in the biosynthesis of a unique polyketide in actinomycetes” Actimomycetologica (2017) 31 [1], S42-S46.

College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan

S47 60th Regular Colloquium

Date: Mar. 10 (Fri), 2017 Place: Kitasato University

Program: 1. “Non-cleaving genome editing and its applications” Keiji NISHIDA (Graduate School of Science, Technology and Innovation, Kobe University)

2. “Bioinformatics for microbial analysis” Wataru IWASAKI (Graduate School of Science, The University of Tokyo)

3.“ Actinomycetaceae showing pathogenicity in animals” Satoshi MURAKAMI (Tokyo University of Agriculture)

4.“Boron-based drug discovery - with the experience of two FDA approvals in a US biotech start-up -” Tsutomu AKAMA ((Former) Anacor Pharmaceuticals, Inc.)

5.“Retraction of papers in major scientific journals; Can you believe what described in the paper? ” Hiroyuki OSADA (RIKEN Center for Sustainable Resource Science)

S48 The 2017 Annual Meeting of the Society for Actinomycetes Japan

Chair person: Masakazu Kataoka (Shinshu University, Nagano)

The 2017 annual meeting of SAJ will be held in September 2017 in Nagano, Japan. We are looking forward to welcoming you to participate in the meeting and to submit papers. Updated information will be provided on the 2017 Annual Meeting Website ( http://awamori.urdr.weblife.me/index.html ) and SAJ Website (http://www.actino.jp/index- e.html).

General Outline

Dates: September 7 (Thu)-­‐8 (Fri), 2017 Venue: The Nagano Wakasato Bunka Hall (http://www.nagano-mwave.co.jp/wakasato_hall/) Address: Wakasato 3-22-2, Nagano 380-0928, Japan TEL: +81-26-223-2223

Registration fee (including abstracts): SAJ member 10,000 yen (8,000 yen until July 14, 2017) Student 3,000 yen (2,000 yen until J July 14, 2017) Non-member 12,000 yen (10,000 yen until July 14, 2017) Non-member Student 4,000 yen (3,000 yen until July 14, 2017) Abstracts only 2,000 yen Registration is acceptable through the 2017 Annual Meeting Website. If you need help, do not hesitate to tell through [email protected]).

Reception: From 19:00 on September 7 (Thu) 2017 at Hotel Metropolitan Nagano, 3F Room Asama (http://www.metro-n.co.jp). SAJ member 9,000 yen (7,000 yen until July 14, 2017) Student 5,000 yen (4,000 yen until July 14, 2017) Non-member 11,000 yen (9,000 yen until July 14, 2017) Non-member Student 8,000 yen (6,000 yen until July 14, 2017)

Scientific program: Invited lectures, SAJ award lectures, and contributed paper sessions (oral/poster) will be arranged.

Submission of abstracts: Abstracts for contributed paper sessions should be submitted via Web-Resister system through Annual Meeting Website as an attachment file. Deadline for submission of abstracts will be on 7th July (Tanabata), 2017.

For further information, contact to: SAJ2016 congress office, c/o Kataoka Lab. Fac. Engineering, Shinshu University Wakasato 4-17-1, Nagano 380-8553, Nagano, Japan Tel: +81-26-269-5538, FAX: +81-26-269-5550 E-mail: [email protected]

S49 Online access to The Journal of Antibiotics for SAJ members

Eligible members of SAJ can access to online issues of The Journal of Antibiotics (JA) by taking following steps;

1. Open the SAJ official website (URL: http://www.actino.jp/) and click the banner of JA. 2. To register, enter your Membership number (10-digit figures starting with 154), First name, Last name, and E-mail address to receive a password and click 'Send'. You can find your Membership number on the envelope from SAJ. 3. Then, you will receive your password from SAJ. 4. Open the SAJ official website (URL: http://www.actino.jp/) and click the banner of JA again. To access the JA website, enter Membership number and password and click 'Login'. 5. Upon recognition of Membership number and password, SAJ site relays the access to the journal's website on nature.com 6. In the journal's website on nature.com, contents are freely available. Members can find the article from current issue table of contents, or archive issues list. Click 'PDF' or 'HTML' link of each article to read full contents.

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S50

日本放線菌学会誌

会 報

第 31 巻 1 号

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受賞論文掲載のおしらせ

2016 年度学会賞受賞 上田 賢志 博士 (日本大学生物資源科学部応用生物科学科)

「放線菌の形態分化と二次代謝の適応応答機構に関する研究」

“Mechanism of adaptive response controlling morphological and physiological development in Streptomyces”

Dr. Kenji UEDA

日本放線菌学会誌 (2017) 31 [1], 2-17.

1 2016 年度日本放線菌学会・学会賞受賞総説

放線菌の形態分化と二次代謝の適応応答機構に関する研究

上田 賢志

日本大学生物資源科学部応用生物科学科 〒252-0880 神奈川県藤沢市亀井野 1866

Mechanism of adaptive response controlling morphological and physiological development in Streptomyces

Kenji Ueda

Department of Applied Biological Science, College of Bioresource Sciences, Nihon University 1866 Kameino, Fujisawa, Kanagawa 252-0880

1.はじめに の増殖を抑える物質」と定義されて今に至 Streptomyces 属に代表される糸状性放線 るが、抗生物質としての活性を示す物質も 菌は、カビに類似した複雑な生活環と抗生 自然界においては抗生物質とは異なる作用 物質をはじめとする様々な二次代謝産物を を有している可能性があることを、ワクス 生産する能力のために、生命科学分野にお マン自身が指摘している 1。 ける基礎と応用の両側面から格好の研究対 筆者は、本総説の前半で解説する A-ファ 象として注目されてきた。特に、この菌群が クターの作用に関する研究から出発して、特 生産する抗生物質ならびに各種生理活性物 にこの菌群の形態分化と二次代謝の開始に 質は医療分野に大きな変革をもたらし、人 影響する要因に着目した研究を進めること 類の生活に欠かせないものになっている。 で、放線菌の複雑な増殖相が多様な環境因子 一方、昨今では、放線菌が生産する代謝産物 に応答して制御されていること、さらには、 が自然界で担っているであろう本来の役割 そうして生産される様々な代謝産物が微生 についての議論も活発になっている。抗生 物コミュニティーの構築に関与し、生態系を 物質(antibiotics)という用語は、ストレ 形作る一つの基盤になっているとする概念 プトマイシンの発見者であるワクスマンに を確立しつつある。ここでは、その道のりか よって「微生物によって生産される微生物 ら最近の知見に至るまでを紹介する。

2

2.自己調節因子、A-ファクター 2-1 生産現場の問題がもたらした再発見 Streptomyces 属の抗生物質生産がγ-ラ クトン骨格を有する自己調節物質(図 1)に よって誘導されることは、今では広く知ら れるようになった。一方、その先駆けとなっ た研究が、ストレプトマイシン(Sm)の生産 現場に於ける生産菌 Streptomyces griseus の遺伝的不安定性に関する観察から始まっ た史実は、今日の溢れる情報の中に埋もれ つつあるかもしれない。 Sm 生産性を失った変異株が高い頻度で出 現するという問題に端を発するその研究は、 東京大学農学部・別府研究室に研究生とし て出向した明治製菓の原によって開始され た。生合成経路中のどこかに変異が起きて いる可能性が想定されたことから、寒天培 地上に異なる変異株を隣接して植え、一方 の株から放出される代謝産物を受け取るこ とでもう一方の株の Sm 合成能が回復する かを観察する cosynthesis 試験が行われた。 図 1 γ-ラクトン型シグナルの例 その結果としてわかったことは、Sm 非生産 A-ファクターは S. griseus の気中菌糸形成と 変異体が失っていたのは Sm 生合成能では 種々の二次代謝産物の生産に、VB-A(バージニ アブタノライドの一誘導体)は S. virginiae の なく、Sm 生産を誘発する拡散性の信号物質 バージニアマイシン生産に、 SCB1 は S. の合成能である、ということであった。 coelicolor A3(2)のアクチノロージン生産にそ れぞれ関与する誘導因子として同定された。 後に A-ファクター(自己調節因子 auto- regulatory factor の略;図1)として広く 知られることになったその信号物質は、そ 記述されている。その後、堀之内によって の時すでにソビエト連邦共和国(当時)の有 A-ファクターの合成遺伝子 afsA が同定さ 機化学者・ココロフによって Sm 合成を誘導 れると 3、その脱落が上記の変異体出現の遺 する分子として単離同定されていた化学 伝的要因であることが明確になった。S. 物質であった。原・別府らが自らの成果を griseus の線状ゲノム上で afsA が位置する A-ファクターの「再発見」と表現したのはそ 末端近傍は脱落が起こりやすく、それが高 のためである。A-ファクター再発見への道 い頻度で Sm 非生産性の変異体が出現する のりについては、別府による総説 2 に詳しく ことの理由として理解されている。

3 図 2 A-ファクターによる気中菌糸形成と Sm 生産の誘発 写真は A-ファクター合成能を欠損した変異株の 6 個のコロニーを A-ファクターの濃度勾配のもと に増殖させた様子。左端の濾紙ディスクに合成 A-ファクターが添加されている。気中菌糸と胞子を 形成しているコロニーは白く、基底菌糸のみを形成しているコロニーは褐色に見える。Sm 生産は一 面に重層した枯草菌に対する増殖阻止ゾーンの大きさによって観察している。

このように、実生産の現場における観察 に結合する蛋白質(受容体)が存在し、(ii) によって、この種のバクテリアに自らの生 それが A-ファクターを受容することで分化 活環をコントロールする分子メカニズムが と二次代謝の両方を制御するマスターレギ 存在することが明らかとなり、産業上極め ュレーター(複数の遺伝子を一斉調節する て重要な本菌群が有する複雑な遺伝生理学 制御蛋白質)のスイッチを入れ、(iii)マス 的特性を掘り下げる基礎研究への入り口が ターレギュレーターは、次に経路特異的レ 開かれた。このような制御は放線菌のみな ギュレーター(各経路の発現調節を専門に らず、グラム陰性のバクテリア集団におい 受け持つ制御蛋白質)のスイッチを入れ、そ ても N-アシルホモセリンラクトン(AHL)の の延長線上でそれぞれの形質の発現が誘導 作用を通じて作動していることが知られて されるという、いわゆるカスケード制御系 いる(クオラムセンシング)。この AHL につ (一つの信号が増幅されながら伝達される いての知見は、発光性のイカに共生する ことで、その制御の下流において様々な機 Vibrio 属細菌のルシフェラーゼ生産制御に 能が一斉に誘発される制御体系)による調 関する基礎遺伝学的研究にはじまるが、興 節がなされていることを推測させた。その 味深いことに、AHL の合成に関与する遺伝子 全貌解明を目標にかかげた堀之内と別府は、 luxI が発見されたのは、上記の堀之内によ ①A-ファクター受容体の同定、および ② る afsA の同定がなされたのと同じ 1984 年 Sm 生合成遺伝子クラスターに A-ファクタ のことであった 4。 ーの信号を伝える制御因子の同定、に的を 絞った。前者は制御の進行方向(図2右図の 2-2 A-ファクターカスケード解明の糸口 上から下の向き)と同一方向に、一方後者は 図2左に示すように、A-ファクターは S. それと逆方向に解析を進めるもので、両者 griseus における Sm 生産と同時に基底菌糸 がつながることで A-ファクターカスケード (栄養細胞)から気中菌糸への形態分化も の中心部が明らかにできると考えたのであ 誘導する。この現象は、(i) A-ファクター る。

4 図 3 Sm 合成遺伝子クラスター と strR プロモーター活性 中央やや左に存在する strR は Sm 合成遺伝子クラスターの初期の 発現を担う正の転写調節因子 を、その下流に同じ向きに隣接 して存在する aphD は Sm 耐性酵 素をコードしている。strR のコ ード領域上流に存在するプロモ ーター領域を切り縮めて A-ファ クター依存性を検定した結果の 概略を併せて表示した。

A-ファクター受容体の同定に至る研究の 推測された。そこで、strR 上流のプロモー 詳細についてはここでは触れないが、大阪 ター活性を、独自に確立した耐熱性リンゴ 大学(当時)の岡本らによって Streptomyces 酸脱水素酵素をレポーターに用いた転写ア virginiae が生産するγ-ラクトンシグナル ッセイによって測定した。上述の A-ファク であるバージニアブタノライド(VB;図1) ター生産性を失った株に人工合成した A-フ の受容体が明らかにされたこと 5 がその礎 ァクターを添加した場合と添加しない場合 になった。岡本は、放射合成されたバージニ でプロモーター活性を比較したところ、 アブタノライドに対する結合活性を指標に strR の転写開始点を含むおよそ 600 bp の 同蛋白質を精製し、そのアミノ酸配列をも 領域に認められる転写活性が A-ファクター とにして遺伝子をクローン化した。その後、 の添加によって上昇することが観察された。 東京大学の尾仲が同様の手法を用いること そこで、その領域をエキソヌクレアーゼを で A-ファクター受容体 ArpA(A-factor- 用いて段階的に切り縮めて同様のアッセイ receptor protein)を単離精製し、遺伝子 を行ったところ、-430 bp までは A-ファク をクローン化した 6。 ターによる誘導性が認められたが、-330 bp 一方、筆者は A-ファクターの信号が Sm 生 まで削るとそれが消失した(図3)。このこ 合成遺伝子クラスターのどこに伝えられる とから、-430 bp から-330 bp の間に A-フ かについての取組みを進めた。Sm 生合成遺 ァクターの信号を伝える転写調節蛋白質が 伝子クラスターは、独のピーパースバーグ 結合すると予想された 8。同蛋白質はヴャク らによって同定され、その塩基配列の解読 リャによってその存在が確認され 9、後に大 がなされていた 7。クラスターの中央付近に 西によって単離精製と遺伝子のクローン化 見いだされた strR 遺伝子は転写調節蛋白 がなされた。adpA ( A-factor-dependent 質をコードしており、耐性酵素遺伝子 aphD protein)と名付けられたこの遺伝子のプロ と隣接した配置にあった(図3)ことから、 モーター領域にはさらに、上記の A-ファ それがクラスターの初期発現に関わる経路 クター受容体である ArpA が結合すること 特異的レギュレーターをコードしていると が判明した 10。

5 図 4 A-ファクターカスケード A-ファクターの信号が受信されることで誘発されるカスケード制御系の模式図。大西博士の好意によ り文献 12 を改編して掲載した。

こうした経緯をもとに、堀之内・大西らに ァクターの作用に関する一連の研究成果は、 よって全貌が明らかにされた A-ファクター 放線菌の複雑な生活環が、精巧に構築され カスケードの概要を図4に示す。上述のよ たカスケード制御系によってプログラムさ うに、strR のプロモーターに結合する活性 れていることを明らかにした貴重な具体事 を手がかりに同定された AdpA は、二次代謝 例となった 11,12。 と形態分化の各経路に特異的な調節遺伝子 群のスイッチを一斉に入れるマスターレギ 2-3 分化誘導ペプチド AmfS ュレーターとして機能し、A-ファクターの A-ファクターの生産能を欠損した上述の 生産以前はその転写が ArpA によって抑制 変異株は、Sm 生産性と同時に気中菌糸を形 されている。A-ファクターが生産されると、 成する能力も失っていたことから、A-ファ それが ArpA に結合して adpA に対する転写 クターカスケードの下には、Sm 生合成遺伝 抑制を解除することでその発現を誘導、二 子群とは独立に、基底菌糸から気中菌糸へ 次代謝産物群の生産と栄養菌糸から気中菌 の細胞分化の開始を制御する遺伝子も含ま 糸・胞子鎖への細胞分化を開始させる。A-フ れると考えられた。細胞分化のメカニズム

6 の 理 解 が 最 も 進 ん で い る Streptomyces する一連の制御遺伝子が存在し、A-ファク coelicolor A3(2)をモデルに用いた研究 13 ターカスケードに連動していると予想され では、基底菌糸から気中菌糸への分化の開 た。 始が起こらなくなった変異株を bld 変異と 上述の研究において、A-ファクター受容 呼び、取得された種々の bld 変異の間の制 体の同定に取り組んでいた三宅は、その遺 御的な上下関係(bld ヒエラルヒー)を、分 伝子クローニングを目的としたショットガ 化 誘 導 ペ プ チ ド SapB ( sporulation- ンクローニングを数多く実施していた。そ associated protein B)の生産性を指標に の過程で同氏は、上述の A-ファクター合成 して推定、整列化する試みがなされた。さら 能欠損変異株にコピー数の高いプラスミド に、遺伝的相補試験などによって各変異に に連結して導入することでその気中菌糸形 対応する bld 遺伝子を特定することで、分 成を回復させる性質をもつ DNA 断片をクロ 化を開始するための一連の制御ネットワー ーン化していた。しかし、この断片の導入は クが明らかにできると考えられた。このア Sm 生産性は回復させなかったことから、そ プローチは、枯草菌 Bacillus subtilis の こには目的の受容体遺伝子とは異なる、細 内生胞子形成の遺伝制御機構に関する研究 胞形態の分化に特異的な役割を持った遺伝 を成功に導いたもので、放線菌においても 子が存在すると考えられた。 その有効性が期待された。S. griseus にも そこで筆者は、三宅によってクローン化 S. coelicolor A3(2)の bld 遺伝子群に対応 された断片の中でも気中菌糸形成の誘導に

図 5 気中菌糸形成誘導ペプチド AmfS の生成メ カニズム A. amf 遺伝子クラスターの発現制御。正の転写 調節因子である AmfR 蛋白質によって amfTSBA オ ペロンの転写が誘導され、前駆体ペプチド AmfS が生成する。本ペプチドはその後 AmfT による修 飾を経て活性型に変換され、膜輸送体 AmfA/AmfB により分泌される。活性型 AmfS は栄養菌糸の表 面に作用することで表面張力を変化させ、気中 菌糸の伸長を促すと考えられている。B. 活性型 AmfS の推定構造。前駆体ペプチドの C-末端側 22 アミノ酸領域中に2つのランチオニン(アラニ ン 2 分子がチオエーテル結合した構造)と2つ のデヒドロアラニン(Dha)が形成され、疎水性 アミノ酸が連続する2つの環状構造をとると予 想されている。C. 活性型 AmfS ペプチドによる 気中菌糸形成の誘発。全面に増殖させた amfS 遺 伝子破壊株は気中菌糸形成を形成する能力が欠 損しているが、活性型 AmfS を添加することでそ の回復がおこる(中央)。

7 重要と考えられた 5.4 kb の塩基配列を決定 作用によって amfTSBA オペロンの転写が活 し、3 つの完全なコード領域を見いだした。 性化される(図5A)。おそらく AmfT が関 そのうちの 2 つは、互いに相同な排出型の 与する修飾(図5Bも参照)を経て前駆体 ABC トランスポーターAmfA と AmfB を、もう AmfS(上述の ORF6 産物に相当)から活性型 ひとつの逆向きのコード領域は二成分制御 ペプチドが生成し、排出ポンプ AmfA・AmfB 系の応答蛋白に相同な転写調節蛋白質 AmfR によって細胞外に輸送され、細胞表層に作 をコードしていた。当時、分化の遺伝制御に 用することで気中菌糸の伸長が誘起される。 関する詳細な理解が進んでいた枯草菌では、 疎水性のアミノ酸配列が露出した球状の構 このファミリーの転写調節蛋白質がそのリ 造をとると推測される活性型 AmfS は、細胞 ン酸化を介して信号を伝達し分化の開始を 表層の表面張力を変化させることで気中菌 決定する役割を担うことが明らかになりつ 糸の伸長を促すと予想されている。amf クラ つあったことから、AmfR が分化の開始に必 スターは S. coelicolor A3(2)にも存在し、 要な遺伝子の転写を誘導する主役であると それによって生成する活性型 AmfS ペプチ 推測された 14。 ドのホモログが、本菌をモデルとした一連 一方で筆者は、上記の領域中にもうひと の形態分化の研究で SapB として知られて つ、43 アミノ酸から成るペプチドをコード きた気中菌糸形成を誘発するペプチドその しうる領域が存在することに気付いていた。 ものであることが明らかに なってい る それが実際に転写・翻訳されるかについて 13,15,16。 の確証はなかったが、上記の排出ポンプを コードする遺伝子の前に存在していたこと、 3. 放線菌代謝産物に見る生物間相互作 ならびに GC-plot(ゲノム中の総 GC 含量が 用 高い菌はアミノ酸を指定するコドンの第三 上述の A-ファクターならびに関連の化合 文字に GC が多く第二文字に少ない傾向が 物は、生産菌自身の二次代謝産物生産なら あることを利用して蛋白質コード領域を推 定するコンピュータープログラム)による 解析がコード領域としての可能性を支持し ていたことから、細胞外に輸送されるペプ チドの可能性を想定して ORF6 として記述 した 14。これが後に、長年にわたって本体が 不明であった S. coelicolor A3(2)の分化 図 6 異種放線菌間のクロストークによる抗 誘導ペプチド SapB の実体解明につながっ 生物質生産の誘発 た。 S. scabrisporus(左端)のコロニーから放 現段階の理解を図5に示す。上述のマス 出される化学物質の濃度勾配に応じて隣接 する S. griseorubiginosus のコロニーにお ターレギュレーターAdpA が直接プロモータ ける抗生物質生産の誘発が認められる。抗生 ーに結合することで転写誘導される amfR 物質は図2と同様に枯草菌の重層によって は転写調節蛋白質をコードしており、その 可視化している。

8 びに形態分化の開始を制御する自己調節因 促進する活性に基づいて単離された 18。デ 子として機能していた。筆者らは次に、類似 フェリオキサミンは、鉄イオンを包み込ん の誘導現象が、異なる放線菌の間において で細胞内に取り込む籠としての役割を有す 観察される可能性について、様々な株の間 るシデロフォアの一種であり、放線菌をは におけるクロストーク実験を通じて検証し じめいくつかの細菌によって生産されるこ た。その結果、予想を上回る数の組み合わせ とが知られている。恐らく、上述の S. において、気中菌糸形成や抗生物質生産の tanashiensis はその生産性を欠損している 促進がおこるものが見いだされた(図6に ために鉄の取り込み効率が低く単独では増 事例)17。それらの結果をもとに、放線菌の 殖能が弱いが、隣接する S. griseus によっ 代謝産物の中には異なる菌の間の相互作用 て生産された同物質が鉄イオンを包摂した を仲介する役割を担うものが含まれる可能 状態で存在すると、それを取り込むことで 性があることを見いだしつつある。 鉄イオンを充足させ、顕著な増殖と分化を 行うと考えられた。 3-1 鉄イオンの奪い合い この S. tanashiensis のように、デフェ 異なる放線菌の種間におけるクロストー リオキサミンの生産能はもたないが、その ク実験をもとに同定された化合物の一つは、 取り込みに関与する膜輸送系を有している デフェリオキサミンE(図7)であった。本 菌は、他にも酵母等で知られていた 18。そこ 化合物は、S. griseus によって生産・分泌 で筆者らはさらに、本化合物の添加によっ され、隣接する Streptomyces tanashiensis て影響を受ける菌株の探索を実施し、デフ の増殖と分化および抗生物質生産を顕著に ェリオキサミンの添加がさまざまな株に多

図 7 異種放線菌間のクロスト ークに基づいて同定された化合 物 デフェリオキサミン E は S. griseus が 生 産 し S. tanashiensis の増殖と分化を誘 発する物質として同定された。プ ロモマイシンは図 6 に示したク ロストークに介在する誘発因子 として、SF2768 はそれによって 生産が誘発される抗生物質とし てそれぞれ同定された。

9 様な形質の変化をひきおこすことを見いだ 一方、異なるシデロフォアについても類似 した 19。特に、Micrococcus 属に属する分離 の群集構造が存在し、それは植物について 株は、デフェリオキサミン添加条件では通 も同様と推測される。このように、環境中に 常の増殖を示したが、非添加の条件ではほ はシデロフォアの利用性にもとづいた複数 とんど増殖が見られないという高い依存性 の集団が存在し、その間の競合と集団内で を示した。また、増殖度には大きく影響しな の協調が環境中における微生物群集の構造 いが、粘性物質や色素の生産、または遊走性 基盤を形成していると考えられる。 が顕著に誘発されるものも見いだされた。 一方、0.1 mM の本物質の添加によって逆に 3-2. 低濃度の抗生物質が誘発する抗生 完全に増殖が阻害されるものも数多く見つ 物質生産-その1 かった。これらの株は、異なるタイプのシデ デフェリオキサミンに続いて筆者らが同 ロフォアに鉄の取り込みを依存し、デフェ 定 し た 化 合 物 は 、 Streptomyces リオキサミンの取り込み能を有しないと推 scabrisporus に分類される分離株によって 測された。そのため、添加された過剰量のデ 生産され Streptomyces griseorubiginosus フェリオキサミンによって鉄がキレートさ の抗生物質生産を誘導する活性(図6)を示 れることで、本来の取り込みメカニズムに す物質である。構造決定の結果、それは図7 よる鉄の獲得ができず、増殖を阻害された に示すポリエーテルであることが明らかに ものと考えられた。 なった 20。プロモマイシンと命名された本 上記の観察は、図8に示すような鉄の取 化合物は、それ自身がイオノフォアとして り込みに関する微生物間の協調と競合の構 作用する抗生物質である。本物質の添加は、 図を想起させる。すなわち、Streptomyces 属 S. griseorubiginosus 以 外 に も 複 数 の は広くデフェリオキサミンを生産・分泌し Streptomyces 属細菌株の抗生物質生産を促 て鉄イオンを包摂して取り込み、活性に鉄 進した。また、モネンシンをはじめとする類 を必要とする酵素等に供給している。さら 似のイオノフォア系抗生物質も同様の活性 にデフェリオキサミンは、Streptomyces 属 を示すことが見いだされた。 以外にもその合成能をもたない株を含め 興味深いことに、プロモマイシンやモネ 様々な菌にも利用されていると考えられる。 図 8 シデロフォアの利用 性に基づく微生物群集構築 放線菌が広く生産するシデ ロフォアであるデフェリオ キサミンは、その生産能力 を持たない他の微生物にも 広く取り込まれ鉄の補給に 利用される。同様の体系は 異なるタイプのシデロフォ アについても存在し、各シ デロフォアグループ間で鉄 の獲得を巡る競合がおこっ ていると考えられる。

10 ンシンによる抗生物質生産の誘導は、それ 遺伝・生理学的背景が異なるものと考えら が抗生物質としての活性を示すより低い濃 れた。上記の化合物は既知であったが、同様 度(subinhibitory concentration of の促進現象によって生産される化合物の中 antibiotics; SICA)においても認められた。 にはこれまでに知られていない構造や作用 昨今では、抗生物質としての活性を示す生 を有するものが含まれる可能性は依然とし 理活性物質も低濃度においてはそれと異な て高く残されていると筆者は考えている。 る生理作用を発揮する可能性が広く議論さ れるようになったが、プロモマイシンの作 3-3 低濃度の抗生物質が誘発する抗生物 用はそれを具体的に示す一つの先駆的事例 質生産-その 2 といえる。 筆者らは、放線菌種間における相互作用 上述の促進現象において、プロモマイシ の探索研究とは独立に、培地への銅イオン ンの作用によって生産が誘発された S. とグルコースの添加が Streptomyces の分 griseorubiginosus の抗生物質は、本菌の単 化と二次代謝に及ぼす影響に着目した研究 独培養では生産がおこらないことから、 を進めた 22,23。特に、低濃度(数μM)の銅 従来の探索では見いだされない新規の化合 イオンの添加は分化と二次代謝の開始に対 物である可能性が期待された。そこで、市販 して促進的に作用することが様々な菌株に のモネンシンを添加した培地条件で生産誘 共通して観察された。このことから、銅イオ 導させた本抗生物質を単離精製し構造解析 ンに依存した制御メカニズムが放線菌にお を行ったところ、本化合物は特許において ける分化の開始に共通して作用しているも 記載がなされているイソニトリル抗生物質 のと予想された。 SF2768 と同一であることが判明した 21。特 そこでまず、SenC/Sco1 ファミリーの蛋白 許に記載の生産菌は単独で本化合物を生産 質に着目した検証を行った 24。このファミ していることから、同一の構造をもつ代謝

産物も菌株によってその生産性を決定する 図 9 銅イオンの取り込みに関与す る sco オペロンの役割 SenC/Sco1 ファミリーの銅シャペロ ンをコードする scoC 遺伝子が含まれ る sco オペロンは Streptomyces 属に 広く分布する。銅イオン欠乏下で転 写が誘導される本オペロンは、銅イ オンの細胞内への取り込みと銅酵素 への運搬に関与していると考えられ る。銅を要求する酵素にはシトクロ ム c 酸化酵素 CcO、リジルオキシダー ゼ HyaS ならびにラッカーゼ EpoA が 含まれる。HyaS は細胞の凝集に、EpoA はリグニンに含まれるフェノールの 酸化に関与する。CcO が高い活性を示 すことは栄養細胞が分化を開始する ために重要であると考えられてい る。

11 リーの蛋白質は多くの微生物に分布し、銅 素シトクロム c オキシダーゼ(CcO)、リジ 要求性の酵素に銅を運搬する銅シャペロン ルオキシダーゼと予想される HyaS、および として機能する。Streptomyces 属放線菌の 小型ラッカーゼ EpoA 25,26 の活性が顕著に低 SenC/Sco1 ホモログは、7 つのコード領域か 下しており、いずれも銅の添加によって回 ら成る sco オペロンの 3 つめの読み取り枠 復することが観察された。このことから、少 scoC にコードされていた(図 9)。本オペロ なくともこれらの酸化酵素への銅の運搬が ンの構造は Streptomyces 属で広く保存さ ScoC によって仲介されていると考えられた れていることから、ここにコード化されて (図 9)。 いる銅イオンの利用に関する機能はこのグ そこで次に、シトクロム c オキシダーゼ ループのバクテリアに共通した生理的意義 をコードする遺伝子の破壊を試みた 27。 をもつものと推測される。 Streptomyces 属放線菌は、多くの好気性細 S. coelicolor A3(2)および S. griseus 菌と同様に 2 種の末端呼吸酵素、シトクロ において scoC を破壊したところ、いずれの ム c オキシダーゼおよびシトクロム bd オキ 破壊株も抗生物質生産と形態分化を行う能 シダーゼを有している(図 10 )。 S. 力の低下を示し、それは銅イオンの添加に coelicolor A3(2)において前者をコードす よって回復した。この結果から、ScoC によ る cta ならびに後者をコードする cyd 遺伝 る銅イオンの運搬に活性を依存する何らか 子をそれぞれ破壊したところ、いずれの破 の銅蛋白質の機能が二次代謝と分化の開始 壊株においても二次代謝と形態分化の能力 に関与していると予想された 24。scoC 破壊 の低下が認められ、特に cta 破壊株でそれ 株では、銅オキシダーゼである末端呼吸酵 が顕著であった。

図 10 放線菌が保有する 2 種の末端呼吸酵素 多くの好気性細菌と同様に、放線菌には 2 種のシトクロム酸化酵素が存在する。銅を要求するシトク ロム c 酸化酵素は酸素分子に対する親和性が低いために高い濃度の酸素を必要とする。一方、銅を必 要としないシトクロム bd 酸化酵素は酸素親和性が高く低い濃度の酸素条件でも活性を示す。いずれの 酸化酵素も破壊すると分化の阻害が観察される。

12 起させた。そこで、破壊株の形質に対する ATP 合成阻害剤 CCCP の添加効果を観察した ところ、増殖を阻害する濃度より低い一定 の濃度範囲において抗生物質生産と気中菌 糸形成の回復が観察された(図 11)27。同様 の効果はオリゴマイシンなどの ATP 合成阻 害剤にも観察された。この現象は、細胞内 ATP レベルが分化・二次代謝の開始に制御的 に連動しているとする仮説を強く支持する ものである。同時に、ATP 合成の阻害剤とし

図 11 ATP 合成阻害剤 CCCP による分化と抗生 て作用する抗生物質にも、上記のイオノフ 物質生産の回復 ォアと同様に、増殖阻害濃度より低濃度で シトクロム c 酸化酵素の銅結合ドメインをコー ドする ctaCD の欠失変異株は通常では気中菌糸 は分化や二次代謝を誘発する因子としての と胞子の形成を行わず、また色素性抗生物質も 作用を発揮するものがあることを強く示唆 生産しないが、ATP 合成阻害剤 CCCP(中央の濾 している。 紙ディスクから供給)の一定の濃度範囲におい てそれらの回復が観察される。 4. 放線菌代謝産物の多様な役割 呼吸酵素の破壊は細胞内のエネルギー準 上記のように筆者らは、放線菌の代謝産 位に大きく影響すると考えられたことから、 物が果たす役割には、従来観察されてきた 次に細胞内の ATP 含量を測定したところ、 抗菌性などの生理活性にとどまらない多様 意外なことに呼吸酵素の破壊株は親株に比 性が存在する可能性を見いだした(図12)。 べて顕著に高い細胞内 ATP レベルを示すこ 一連の研究の引き金を引いた A-ファクター とが判明した 27。顕著に高い細胞内 ATP 含 は、生産菌自身に作用してその二次代謝な 量は、上記の scoC 破壊株およびいくつかの らびに形態分化の開始を誘発するスイッチ bld 変異株においても同様に観察された。呼 として機能し、同一集団内の細胞が同じタ 吸酵素の破壊がなぜ ATP レベルの上昇を引 イミングで抗生物質生産と形態分化を開始 き起こすかは現在不明であるが、呼吸欠損 することを可能にしていた。生産菌自身の を補う恒常性維持機構が発動することで誘 増殖も阻害する抗生物質の場合、その生産 発される代替の代謝メカニズムが ATP の生 に先立ってあらかじめそれに対する耐性メ 成レベルを維持すると同時に、ATP を消費す カニズムを発現させておく必要がある。そ る効率が何らかの要因によって低下するこ のため、上記の Sm の事例に見られるように、 とがその背景にあるものと考えられる。 抗生物質の生合成クラスター中には初期に 上記の呼吸酵素欠損株についての観察は、 発現する遺伝子群の中に自己耐性遺伝子が エネルギー代謝と分化の開始が連携してお 含まれていることが多い。しかし、そのクラ り、細胞内 ATP レベルが分化開始に対する スターの発現時期が細胞集団全体でそろわ 信号としての役割を担っている可能性を想 ないと、一部の細胞によって生産された抗

13 AHL についてもその合成遺伝子と受容体遺 伝子のゲノム上の分布に基づいた議論がな されている 29。 異なる放線菌の間においてクロストーク が実際におこることは、筆者らが実施した 様々な株の間におけるクロストークアッセ

図 12 放線菌の代謝産物が果たす多様な役割(概 イによって強く示唆された。一方の菌が生 念図) 産する拡散性物質に応答してもう一方の菌 放線菌が生産・分泌する代謝産物には、自身の形 の分化や抗生物質生産が促進されていると 質を制御する自己調節因子や鉄をはじめとする環 境中の特定の因子を取り込むための分子が存在す 考えられる組み合わせの中には、γ-ラクト るが、それらの中には他の菌にも同様に受容ない ン化合物による作用が含まれている可能性 し利用されるものが含まれる。また、高濃度の抗 も考えられるが、これまでに同定された因 生物質のように他の菌の増殖に阻害的に作用する もの、一方で同じ抗生物質も低濃度では異なる作 子はいずれもγ-ラクトン化合物とは異な 用を示すものも存在する。そうした複雑な代謝産 る代謝産物であった。 物の作用の重なりの上に微生物の群集構造が構築 されていると考えられる。 種間促進因子として一つ目に同定された デフェリオキサミンは、放線菌によって広 生物質によって周辺の細胞が死滅する可能 く生産されるシデロフォアであった。筆者 性がある。形態分化についても、気中菌糸形 らによる調査は、鉄イオンを包摂した本物 成の開始とともに栄養菌糸を分解するため 質は、その生産能力を持たない菌にも広く に発現するプロテアーゼ等が同様の影響を 取り込まれ、鉄イオン源として利用されて 及ぼす可能性がある。A-ファクターが拡散 いることを示していた。このことから、本物 して一斉に作用することは、そうしたタイ 質は微生物界におけるグローバルな共生因 ミングのずれを回避し、細胞集団を同調的 子としての役割を果たしていると考えるこ に制御するために重要な役割をもつと推測 とができる。一見、生産者の放線菌はエネル される。 ギーを浪費しているように思われるが、本 A-ファクターやバージニアブタノライド シデロフォアを様々な菌が利用することは などのγ-ラクトンシグナルは、その受容体 生産菌の周囲に活発な代謝活性をもつ微生 への結合特異性が厳密で、異なる側鎖構造 物コミュニティーを発達させることにつな を有するγ-ラクトンを作る種の間での交 がり、それが生産菌に副次的なメリットを 信は起こらないと考えられている。一方、同 もたらす可能性も考えられる。 一の構造をもつγ-ラクトンが複数の異な 二つ目に同定されたプロモマイシンは、 る種によって生産される事例も知られてお モネンシンに類縁のポリエーテルで、イオ り、そうした場合には種間クロストークが ノフォアとして作用する抗生物質であった。 起こる可能性が指摘されている 28。同様の 特筆すべき点は、本物質の二次代謝促進活 細菌種内・種間における信号のやりとりの 性が抗生物質として作用するより低い濃度 可能性は、前述のグラム陰性菌が生産する でも認められたことで、抗生物質として知

14 られる化合物が阻害濃度未満において抗生 は、放線菌の二次代謝遺伝子群の多くが通 物質とは異なる生理作用を示す事例となっ 常の培養条件では発現していないという問 た。冒頭で触れたように、抗生物質が自然界 題が大きく取り上げられているが、筆者ら において実際に抗生物質として作用するか の研究成果は、そうした遺伝子の中には他 については、抗生物質を定義したワクスマ 生物との共生・共存・競合を含め、多様な ン自身も疑問を投げかけている。その詳細 環境への適応応答に依存して発現するもの は明らかでないが、上記のポリエーテルに が含まれる可能性を示している。これはす よる抗生物質生産の促進にはイオノフォア なわち、そうした従来の培養条件には反映 としてのそれとは異なる作用機序が関与し されていない要素をいかに分離・培養系に ている可能性がある。 取り入れることができるかが、これからの 阻害濃度未満の抗生物質が放線菌の分化 微生物探索における重要課題であることを と二次代謝を誘発する事例として、筆者ら 意味している 30。 は ATP 合成阻害剤にも同様の効果を見いだ 代謝産物の機能が生物群集構造の構築を した。この効果はおそらく、末端呼吸におけ 担っていることはまた、そうした化合物の る異常によって起こる細胞内 ATP レベルの 作用を通じて目に見えない群集の実態を探 上昇が低濃度の呼吸阻害剤によってある水 る研究手法に一定の有効性を見いだせる可 準にまで抑えられることによってひき起こ 能性を示唆している。農芸化学分野で古く されると考えられる。なぜ呼吸の異常によ から取り組まれてきた、特定化合物をプロ って細胞内 ATP レベルが増大するのかにつ ーブに用いて生体中の複雑な生理現象とそ いての詳細は不明であるが、一次代謝にお の分子メカニズムを探る研究手法は、最近 けるエネルギー効率と二次代謝・細胞分化 ではケミカルバイオロジーという言葉を持 の開始決定機構が制御的に連動しているこ って広く認識されるようになった。同様の とは想像に難くない。そこに細胞内 ATP レ 概念は複雑な生態系の理解にも適用できる ベルが指標として関わっていることは、放 可能性があり、実際にケミカルエコロジー 線菌の生理学的特性ならびに有用物質生産 と呼ばれる生態学研究領域が主として昆虫 に関する基礎的な理解に対し、新たな洞察 フェロモンの作用に着目して発展してき を与えるものと期待される。 た。筆者は、同様の研究手法を通じて、見 えない微生物の群集構造が基礎となる生態 5. おわりに 系にあらたな理解が進むと同時に、そこか 筆者らは、放線菌の形態分化と二次代謝 ら次世代の微生物バイオテクノロジーが拓 が自己調節物質と内在因子によって柔軟か かれることを期待している。 つ精密に調節される一方、その代謝産物は 異なる生物どうしの間の共生相互作用に介 在して多様な作用を発揮し、それらを通じ 謝辞 て生物群集構造の構築に一定の役割を果た 本研究は、主として日本大学生物資源科学 しているという概念を創出した。昨今で 部・生命工学研究室において、またその礎

15 となった研究は東京大学農学部・醗酵学教 7. Distler, J., Ebert, A., Mansouri, K., Pissowotzki, K., Stockmann, M. and Piepersberg, W. (1987). 室において行われました。格別のご指導を Gene cluster for streptomycin biosynthesis in 賜りました別府輝彦先生ならびに故堀之内 Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity. 末治先生に深く感謝申し上げます。同時 Nucleic Acids Res., 15, 8041-8056. に、本研究推進の要所を担ってくださった 8. Vujaklija, D., Ueda, K., Hong, S.K., Beppu, T. and Horinouchi, S. (1991). Identification of an A- 高野英晃博士、白鳥初美博士、天野昭一さ factor-dependent promoter in the streptomycin んをはじめとする生命工学研究室の同僚な biosynthetic gene cluster of Streptomyces griseus. Mol. Gen. Genet., 229, 119-128. らびに卒業生・学生の皆様の多大な努力に 9. Vujaklija, D., Horinouchi, S. and Beppu, T. (1993). 心から敬意と謝意を表します。吉田 稔先 Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, 生、西山 真先生、洪 淳光博士、Duška a regulatory gene for streptomycin biosynthesis in Vujaklija 博士、三宅克英博士、大西康夫 Streptomyces griseus. J. Bacteriol., 175, 2652- 2661. 博士、尾仲宏康博士をはじめとする醗酵学 10. Ohnishi, Y., Kameyama, S., Onaka, H. and 教室時代の指導者・同門の皆様、本研究を Horinouchi, S. (1999). The A-factor regulatory cascade leading to streptomycin biosynthesis in 様々にご指導・ご支援くださいました作田 Streptomyces griseus: identification of a target 庄平先生、降旗一夫先生、池田治生先生、 gene of the A-factor receptor. Mol. Microbiol., 34, 102-111. 岡本晋博士はじめ日本放線菌学会の皆々様 11. Horinouchi, S. (2007). Mining and polishing of the に厚く御礼申し上げます。 treasure trove in the bacterial genus Streptomyces. Biosci. Biotechnol. Biochem., 71, 283-299.

12. Horinouchi, S. and Beppu, T. (2007). Hormonal

control by A-factor of morphological development 引用文献 and secondary metabolism in Streptomyces. Proc. 1. Davies, J. (2006). Are antibiotics naturally Jpn. Acad. Ser. B Phys Biol Sci, 83, 277-295. antibiotics? J. Ind. Microbiol. Biotechnol., 33, 13. Chandra, G. and Chater, K.F. (2014). 496-499. Developmental biology of Streptomyces from the 2. 別府輝彦. (2010). A-ファクターの再発見. 化学 perspective of 100 actinobacterial genome と生物, 48, 498-502. sequences. FEMS Microbiol. Rev., 38, 345-379. 3. Horinouchi, S., Kumada, Y. and Beppu, T. (1984). 14. Ueda, K., Miyake, K., Horinouchi, S. and Beppu, Unstable genetic determinant of A-factor T. (1993). A gene cluster involved in aerial biosynthesis in streptomycin-producing mycelium formation in Streptomyces griseus organisms: cloning and characterization. J. encodes proteins similar to the response regulators Bacteriol., 158, 481-487. of two-component regulatory systems and 4. Engebrecht, J. and Silverman, M. (1984). membrane translocators. J. Bacteriol., 175, 2006- Identification of genes and gene products 2016. necessary for bacterial bioluminescence. Proc. 15. Kodani, S., Hudson, M.E., Durrant, M.C., Natl. Acad. Sci. U S A, 81, 4154-4158. Buttner, M.J., Nodwell, J.R. and Willey, J.M. 5. Okamoto, S., Nakamura, K., Nihira, T. and (2004). The SapB morphogen is a lantibiotic-like Yamada, Y. (1995). Virginiae butanolide binding peptide derived from the product of the protein from Streptomyces virginiae. Evidence that developmental gene ramS in Streptomyces VbrA is not the virginiae butanolide binding coelicolor. Proc. Natl. Acad. Sci. U S A, 101, protein and reidentification of the true binding 11448-11453. protein. J. Biol. Chem., 270, 12319-12326. 16. Takano, H., Matsui, Y., Nomura, J., Fujimoto, M., 6. Onaka, H., Ando, N., Nihira, T., Yamada, Y., Katsumata, N., Koyama, T., Mizuno, I., Amano, Beppu, T. and Horinouchi, S. (1995). Cloning S., Shiratori-Takano, H., Komatsu, M. et al. and characterization of the A-factor receptor gene (2017). High production of a class III lantipeptide from Streptomyces griseus. J. Bacteriol., 177, AmfS in Streptomyces griseus. Biosci. Biotechnol. 6083-6092. Biochem., 81, 153-164.

16 17. Ueda, K., Kawai, S., Ogawa, H., Kiyama, A., differentiation in Streptomyces griseus. Antonie Kubota, T., Kawanobe, H. and Beppu, T. (2000). Van Leeuwenhoek, 78, 263-268. Wide distribution of interspecific stimulatory 24. Fujimoto, M., Yamada, A., Kurosawa, J., Kawata, events on antibiotic production and sporulation A., Beppu, T., Takano, H. and Ueda, K. (2012). among Streptomyces species. J. Antibiot., 53, 979- Pleiotropic role of the Sco1/SenC family copper 982. chaperone in the physiology of Streptomyces. 18. Yamanaka, K., Oikawa, H., Ogawa, H.O., Hosono, Microb. Biotechnol., 5, 477-488. K., Shinmachi, F., Takano, H., Sakuda, S., 25. Endo, K., Hayashi, Y., Hibi, T., Hosono, K., Beppu, Beppu, T. and Ueda, K. (2005). Desferrioxamine T. and Ueda, K. (2003). Enzymological E produced by Streptomyces griseus stimulates characterization of EpoA, a laccase-like phenol growth and development of Streptomyces oxidase produced by Streptomyces griseus. J. tanashiensis. Microbiology, 151, 2899-2905. Biochem., 133, 671-677. 19. Eto, D., Watanabe, K., Saeki, H., Oinuma, K., 26. Endo, K., Hosono, K., Beppu, T. and Ueda, K. Otani, K., Nobukuni, M., Shiratori-Takano, H., (2002). A novel extracytoplasmic phenol oxidase Takano, H., Beppu, T. and Ueda, K. (2013). of Streptomyces: its possible involvement in the Divergent effects of desferrioxamine on bacterial onset of morphogenesis. Microbiology, 148, 1767- growth and characteristics. J. Antibiot., 66, 199- 1776. 203. 27. Fujimoto, M., Chijiwa, M., Nishiyama, T., Takano, 20. Amano, S., Morota, T., Kano, Y.K., Narita, H., H. and Ueda, K. (2016). Developmental defect of Hashidzume, T., Yamamoto, S., Mizutani, K., cytochrome oxidase mutants of Streptomyces Sakuda, S., Furihata, K., Takano-Shiratori, H. coelicolor A3(2).. Microbiology, 162, 1446-1455. et al. (2010). Promomycin, a polyether promoting 28. Nodwell, J.R. (2014). Are you talking to me? A antibiotic production in Streptomyces spp. J. possible role for gamma-butyrolactones in Antibiot., 63, 486-491. interspecies signalling. Mol. Microbiol., 94, 483- 21. Amano, S.I., Sakurai, T., Endo, K., Takano, H., 485. Beppu, T., Furihata, K., Sakuda, S. and Ueda, 29. Subramoni, S. and Venturi, V. (2009). LuxR- K. (2011). A cryptic antibiotic triggered by family 'solos': bachelor sensors/regulators of monensin. J. Antibiot., 64, 703. signalling molecules. Microbiology, 155, 1377- 22. Ueda, K., Tomaru, Y., Endoh, K. and Beppu, T. 1385. (1997). Stimulatory effect of copper on antibiotic 30. Ueda, K. and Beppu, T. (2017). Antibiotics in production and morphological differentiation in microbial coculture. J. Antibiot., 70, 361-365 Streptomyces tanashiensis. J. Antibiot., 50, 693- 695. 23. Ueda, K., Endo, K., Takano, H., Nishimoto, M., Kido, Y., Tomaru, Y., Matsuda, K. and Beppu, T. (2000). Carbon-source-dependent transcripti- onal control involved in the initiation of cellular

17 2016年度学会賞受賞

上田 賢志 博士

(日本大学生物資源科学部)

「放線菌の形態分化と二次代謝の適応応答機構に関する研究」

Dr. Kenji Ueda

Mechanism of adaptive response controlling morphological and physiological development in Streptomyces

Department of Applied Biological Science, College of Bioresource Sciences, Nihon University

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2017 年度(第 32 回)日本放線菌学会大会のご案内

大会長 片岡 正和 (信州大学大学院総合理工学系研究科生命医工学専攻)

2017 年度日本放線菌学会大会は、長野県長野市若里文化ホールにて開催することになりました。 日本を代表する観光地長野でさわやかな気候、おいしい季節の開催となります。 多くの皆様のご参 加を心よりお待ち申し上げます。 詳しい情報は大会のウェブサイト(http://awamori.urdr.weblife.me/index.html)、日本放線菌学会の ウェブサイト(http://www.actino.jp)を通じて随時ご案内いたします。

概 要

期日:平成 29 年 9 月 7 日(木),8 日(金) 会場:長野市若里市民文化ホール 〒380-0928 長野市若里3丁目22番2号 (徒歩またはタクシーをお奨めします) (JR 長野駅徒歩 20 分,タクシー10分,バス 15 分) 長野駅善光寺口(2番のりば)~日赤・松岡線「ビッグハット前」下車徒歩1分 路線番号:21 番「松岡・サンマリーン・大塚南」) TEL:026-223-2223 http://www.nagano-mwave.co.jp/wakasato_hall/

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参加費(講演要旨集代を含む) 7 月 14 日まで 7 月 15 日~当日 正会員: 8,000 円 10,000 円 学生会員: 2,000 円 3,000 円 非会員: 10,000 円 12,000 円 非会員学生: 3,000 円 4,000 円 *要旨集(2,000 円)のみをご希望の方は, 大会事務局までご連絡下さい。

懇親会 日時:平成 29 年 9 月 7 日(木)19:00~21:00 (予定) 会場:ホテルメトロポリタン長野 (http://www.metro-n.co.jp) 3F あさま 会費: 7 月 14 日まで 7 月 15 日~当日 正会員: 7,000 円 9,000 円 学生: 4,000 円 5,000 円 非会員: 9,000 円 11,000 円 非会員学生: 6,000 円 8,000 円

プログラム概要(詳細は大会ウェブサイトをご覧下さい。) 1. 一般講演(口頭発表とポスター発表 [ショートトークあり]) 2. 受賞講演 3. 招待講演 (英国と日本のシステム・合成生物学関連研究者を予定) 4. エクスカーション 大会終了後バスで善光寺へ、18 時頃長野駅着 (無料)

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参加および講演申し込み要領

● 参加および講演申し込み (今大会から大会登録システムを導入しています) 大会ウェブサイト(http://awamori.urdr.weblife.me/index.html)の参加登録より、リンク先の大会登録シ ステムで参加・講演登録して下さい。 *講演申し込み、講演要旨提出の締切日:平成 29 年 7 月 7 日(金)七夕 *大会参加の事前申し込みの締切日:平成 29 年 7 月 14 日(金)

● 参加費・懇親会費等の振込み:下記の口座へお振込み下さい。お振込みには郵便局備え付けの 払込用紙、ATM 払込みもしくは郵貯ダイレクトをご利用下さい。

郵便局から 口座記号番号 :11190 − 37196921 口座名称(漢字):日本放線菌学会第 32 回大会 口座名称(カナ):ニホンホウセンキンガッカイダイサンジュウニカイタイカイ

他行等から 銀行名:ゆうちょ銀行 店名:一一八(イチイチハチ)店、店番:118 預金種目:普通 口座番号:3719692 口座名称(カナ):ニホンホウセンキンガッカイダイサンジュウニカイタイカイ

● 講演要旨:大会登録システムにある雛形をダウンロードして登録システムで入稿して下さい。 所属は和文・英文とも省略形で記載してください。英文タイトル等は英文プログラムに使用し ますので, 2 頁目に記載して下さい。 ● 発表形式の詳細等は、電子メールにてお知らせいたします。 ● 発表スライドならびにポスターは英語で作成することを推奨します。

お問合せ先(大会事務局) 〒380-8553 長野市若里 4-17-1 信州大学工学部 片岡研内 第 32 回放線菌学会大会事務局 Tel: 026-269-5538 E-mail: [email protected]

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2017年度日本放線菌学会三賞授賞者の決定について

2017年4月21日 会長 早川 正幸

日本放線菌学会は、下記のように2017年度日本放線菌学会三賞授賞者を決定しましたので以下にご 報告致します。 日本放線菌学会大村賞(学会賞)および日本放線菌学会功績功労賞候補者については、理事、評議 員、監事およびその経験者が推薦することができます。日本放線菌学会浜田賞(研究奨励賞)候補 者については、自薦も含めてすべての正会員が推薦できることになっておりますので、今後も、積 極的なご推薦をお願い申し上げます。

【大村賞(学会賞)】 田村 朋彦氏(独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター) 「日本及びアジア地域の放線菌多様性の研究とNBRC放線菌リソースの充実」

【功績功労賞】 浅野 行蔵氏 (北海道大学大学院農学研究院名誉教授, 旭川食品産業支援センター センター長) 「希少放線菌属の探索・発見に関する研究および学会への貢献」

【浜田賞(研究奨励賞)】 稲橋 佑起氏(北里大学北里生命科学研究所) 「植物由来放線菌の分離とその応用研究」

以上

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報告 第 60 回日本放線菌学会学術講演会

主催 : 日本放線菌学会 日時 : 平成 29 年 3 月 10 日(金) 13:00~17:40 場所 : 北里大学薬学部 1 号館 6 階 1603 教室 参加者: 64 名

プログラム 1.『切らないゲノム塩基編集の多様な生物への応用』 西田 敬二 (神戸大学 大学院科学技術イノベーション研究科) 2.『微生物解析におけるバイオインフォマティクスの活用』 岩崎 渉 (東京大学 大学院理学系研究科) 3.『動物に病原性を示す放線菌 Actinomycetaceae』 村上 覚史 (東京農業大学 農学部) 4.『ホウ素を元にした創薬 ―米国ベンチャー企業で2剤の FDA 認可を受けた経験談とともにー』 赤間 勉 (元 Anacor Pharmaceuticals,Inc.) 5.『一流誌で目立つ論文撤回:その論文、信じられますか?』 長田 裕之 (理化学研究所 環境資源科学研究センター)

切らないゲノム塩基編集の多様な生物への応用 西田 敬二 (神戸大学大学院科学技術イノベーション研究科) [email protected]

様々な生物種のゲノム情報を直接操作することが出来るゲノム編集技術は近年著しい進歩を遂げ ており、生物工学はもちろんのこと生命科学全域においても革命的なツールとなりつつある。代表的 な ZFN、TALEN、CRISPR などは人工ヌクレアーゼと呼ばれるものであるが、これらの技術はいずれ も標的としたい DNA 配列を特異的に認識するようデザインすることが可能であり、標的部位におい て DNA 二重鎖切断を引き起こして、その後に宿主細胞が修復する過程で配列の変換を期待するもの である。これまで相同組み換えおよびジーンターゲッティングが困難であった材料においても非常に 有効であることから、動物など高等真核生物を中心に急速に導入が進んでいる。しかしながら染色体 切断による細胞毒性が問題となり、特に多くの微生物等では多くの場合に致死的で利用法が限定的と なっている。 私たちは新たなゲノム編集技術として、ヌクレアーゼ活性と代わる脱アミノ化による塩基変換反応 を採用することによって DNA を切らずに書き換える新たなゲノム編集技術 Target-AID の開発に成功

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した。これにより標的に点変異を直接導入してゲノム情報を書き換えることが可能で、より精密でか つ細胞毒性の低いゲノム編集技術として確立することができた。あらゆる生物種において実証が進め られているが、特に微生物においては多点同時変異などが容易に行えるようになり、幅広い応用展開 が期待される。

参考文献 1) Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune sys- tems. Science (2016)

2) Shimatani et al., Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fu- sion. Nature Biotechnology. accepted

微生物解析におけるバイオインフォマティクスの活用 岩崎 渉 (東京大学 大学院理学系研究科) [email protected]

近年の微生物解析においてバイオインフォマティクスは不可欠なものとなった。特に第二世代DN Aシーケンサ(次世代DNAシーケンサ、超並列DNAシーケンサとも)の登場はゲノム・メタゲノ ムデータ取得のスピードを革命的に向上するとともに、そのコストを大幅に押し下げた 1)。急激な技 術革新を表す経験則としてはコンピュータの集積回路に使われる「ムーアの法則」が人口に膾炙して いるが、驚くべきことに、DNAシーケンサの性能向上のスピードはそのムーアの法則によるコンピ ュータの性能向上のスピードを遥かに上回っている。必然的に、第二世代DNAシーケンサから産出 されるデータを「力ずくで」コンピュータ解析するアプローチには限界が見えつつあり、バイオイン フォマティクス分野においては、最新の情報科学のテクニックに基づいた高速なアルゴリズムの開発、 および、新しい生物学的アイデアに基づいた巧妙な切り口からの解析手法の開発が喫緊の課題となっ ている。

DNAシーケンサの性能向上が微生物学分野にもたらした最も大きな変化は、第一に解読済み微生 物ゲノム配列数の加速度的なペースでの増加であり、第二に環境中の微生物ゲノムDNAを直接読み 取るメタゲノム解析の普遍化であろう。世界中で決められつつあるゲノム配列の全貌を把握しうる組 織はすでにどこにも存在しないが、このことは、微生物ゲノム配列データを大規模に比較解析するこ とで、微生物ゲノムがどのように形作られるのか、そのメカニズムを探求することが可能になりつつ あることを意味している。これまでに演者ら自身も、過去の微生物種がどのようなゲノムを持ってい たかを高精度に推定するためのアルゴリズムを開発する 2)とともに、微生物ゲノムの進化の過程で 遺伝子水平伝播や遺伝子・ゲノムの重複が大きな役割を果たしたことを明らかにしてきた 3,4,5)。また、 メタゲノム解析は数年前まではごく限られたプロジェクトにおいて採用される研究アプローチであ

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ったが、現在では一つの研究室単位でも行われる日常的なアプローチとなった 6)。これらのデータが もたらす膨大な環境微生物叢情報により、現在では、微生物と環境との関係をより俯瞰的な視点から 捉えることが可能になりつつある 7)。

本講演では、これらの大量のゲノムデータ・メタゲノムデータに日々接しバイオインフォマティク ス研究を進めている立場から、話題を提供したい。

参考文献 1) Satoshi Hiraoka, Ching-chia Yang, and Wataru Iwasaki. Metagenomics and bioinformatics in microbial ecology: Current status and beyond. Microbes and Envi- ronments, 31, 204-212. (2016) 2) Wataru Iwasaki and Toshihisa Takagi. Reconstruction of highly heterogeneous gene-content evolution across the three domains of life. Bioinfor- matics, 23, i230-i239. (2007) 3) Wataru Iwasaki and Toshihisa Takagi. Rapid pathway evolution facilitated by horizontal gene transfers across prokaryotic lineages. PLOS Ge- netics, 5, e1000402. (2009) 4) Seishiro Aoki, Motomi Ito, and Wataru Iwasaki. From beta- to alpha-proteobacteria: the origin and evolution of rhizobial nodulation genes nodIJ. Molecu- lar Biology and Evolution, 30, 2494-2508. (2013) 5) Sira Sriswasdi, Masako Takashima, Ri-ichiroh Manabe, Moriya Ohkuma, Takashi Sugita, and Wataru Iwasaki. Global deceleration of gene evolution following recent genome hybridizations in fungi. Genome Re- search, 26, 1081-1090. (2016) 6) Satoshi Hiraoka, Asako Machiyama, Minoru Ijichi, Kentaro Inoue, Kenshiro Oshima, Masahira Hattori, Susumu Yoshizawa, Kazuhiro Kogure, and Wataru Iwasaki. Genomic and metagenomic analysis of microbes in a soil environment affected by the 2011 Great East Japan Earthquake Tsunami. BMC Genomics, 17, 53. (2016) 7) Ching-chia Yang and Wataru Iwasaki. MetaMetaDB: A database and analytic system for investigating microbial habitability. PLOS ONE, 9, e87126. (2014)

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動物に病原性を示す放線菌 Actinomycetaceae 村上 覚史 (東京農業大学農学部畜産学科家畜衛生学研究室) [email protected]

獣医師は放線菌と聞くと、誰もが牛の顎に形成される放線菌症の原因菌 Actinomyces bovis を思い浮 かべます。放線菌学会の会員の方々は放線菌と言えば、抗生物質などを生産する Streptomyces 属のこ とを思い浮かべることでしょう。寄生虫の駆虫薬、イベルメクチンを知らない獣医師はいませんが、 大村智先生のノーベル賞受賞で、それが放線菌の産物であったことを知った獣医師も多かったのでは ないかと思います。獣医師や医師は放線菌と言えばイコール放線菌症を思い浮るのです。そこで、今 回特に動物における Actinomycetaceae について、私が経験してきた幾つかの症例をもとに病原体とし ての放線菌についてお話してみようと思います. 臨床的に動物の放線菌症は牛でよく知られ、顎に腫瘍のようなゴツゴツと盛り上がる病巣が形成さ れ、通称”lumpy jaw”と言われていました。その病巣内に放射状に広がる菌塊(ray-fungus of the cow) がみられることから A. bovis という種名が誕生しました。豚においても臨床的な放線菌症は昔から知 られており、牛と同様の病巣が乳房に形成されます。起因菌は Actinomyces suis と名付けられていま したが、しかしその種名である A. suis は現在、尿路感染症の起因菌である Eubacterium suis から転属 された種名として扱われています。したがって、豚の乳房放線菌症の起因菌としての種名ではありま せん。 Actinomyces 属は口腔内常在菌であると一般に考えられていることから牛の口腔で A. bovis の存在 の有無が調べられています。しかし分離された Actinomyces は A. denticolens, A. howellii, A. slackii とい う新種ばかりで、肝心の A. bovis はこれまで分離されていません。ヒトの扁桃では、その陰窩に腐生 的な放線菌塊がよくみられます。動物では豚の扁桃陰窩で普通に放線菌塊が存在し、この菌塊は典型 的な放線菌病巣を形成します。豚扁桃分離株を母豚の乳房に接種すると乳房放線菌症を惹起します。 われわれは、牛に病原性の強い A. bovis も扁桃陰窩で放線菌病巣を形成するはずだと考え、67 頭の口 蓋扁桃を集めて調べましたが、病巣は全く存在しませんでした。最近、海外の馬下顎リンパ節膿瘍か ら A. denticolens が分離されたことから馬の扁桃を調べたところ、ほとんどの扁桃陰窩に放線菌塊が 存在し、それらの分離菌は牛の口腔から分離された A. denticolens と同一菌種でした 1)。さらに驚いた ことには、豚の扁桃で常在し、乳房放線菌症を起こした Actinomyces sp.も A. denticolens と同一種であ ることが判明しました(第 31 回放線菌学会)。 その他、Actinomyces 以外にも Arcanobacterium pyogenes は牛に流産を、豚に皮下膿瘍を起こします が、豚での流産は知られていません。ある時、豚の流産胎仔を調べたところ、その肺病変が牛のそれ とよく類似したことから豚の Arcanobacterium pyogenes による流産かと考えましたが、細菌学的及び 16S rRNA 解析の結果、新種となり Arcanobacterium abortisuis と命名されました 2)。現在、両種は Trueperella という新たな属に転属されています 3)。

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参考文献 1) Azuma, R., Murakami, S., Ogawa, A., Okada, Y., Myazaki, S. and Makino, T., Arcanobacterium abor- tisuis sp. nov., isolated from a placenta of a sow following an abortion. Int. J. Syst. Evol. Microbiol., 59: 1469-1473. (2009) 2) Murakami, S., Otaki, M., Hayashi, Y., Higuchi, K., Kobayashi, T., Torii, Y., Yokoyama, E., and Azuma, R., Actinomyces denticolens colonisation identified in equine tonsillar crypts. Vet Rec Open 8, 3(1):e000161. (2016) 3) Yassin AF, Hupfer H, Siering C. and Schumann P., Comparative chemotaxonomic and phylogenetic stud- ies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. Int J Syst Evol Microbiol., 61:1265- 1274. (2011)

ホウ素を元にした創薬 -米国ベンチャー企業で 2 剤の FDA 認可を受けた経験談とともに- 赤間 勉 ((元)Anacor Pharmaceuticals, Inc.) [email protected]

ホウ素は植物に必須な微量栄養素であり、ヒトは野菜や果物などから、平均して1日数ミリグラ ム程度のホウ素を摂取していると考えられている。1) 天然のホウ素は、主にホウ酸またはそのエス テル類として存在し、炭素-ホウ素結合を有する有機ホウ素化合物は天然からは見つかっていな い。2) 元素周期表上で炭素の左隣に位置するホウ素を含む化合物には、空の p 軌道のルイス酸性のた め、アルコールの水酸基などと相互作用することにより、sp2-sp3 の 2 つのコンフォメーション間 の平衡が存在する。この性質を利用することにより、タンパク質や核酸など様々な創薬ターゲット と相互作用させることができ、従来の炭素ベースのものとは異なる、新規な医薬品の開発につなが ることが期待された。 しかしながら、化学合成された多くの含ホウ素有機化合物が様々な生理活性を示すことは、長年 知られてきたものの、2002 年以前は医薬品として開発されるまでには至らなかった。1) 2003 年に、世界初のホウ素含有医薬品となる bortezmib (Velcade®) が多発性骨髄腫の治療薬(注 射剤)として FDA から認可された。3) その後、2014 年に tavaborole (Kerydin®) が爪白癬の外用剤 として、4) 2015 年に bortezmib の第 2 世代となる ixazomib (Ninlaro®) が経口剤として、5) そして 2016 年に crisaborole (EucrisaTM) がアトピー性皮膚炎の外用剤として FDA から認可される 6)という、 ここ数年の認可ラッシュにより、現在までに 4 剤のホウ素含有医薬品が生まれている。

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OH O OH H B N N B N OH O H O F N Tavaborole Bortezomib

Cl O OH H OH N B N N OH B H O O O Cl Crisaborole Ixazomib

上記 4 剤のうち、tavaborole および crisaborole は、米国カリフォルニア州 Palo Alto に位置する An- acor Pharmaceuticals によって開発された。従来の生理活性有機ホウ素化合物の大部分が脂肪族ホウ 酸誘導体であったのに対して、Anacor 社は芳香環と縮環した benzoxaborole 誘導体に注目して研究開 発を行った。Benzoxaborole 誘導体は、医薬品候補物質として考えた場合、化学的および代謝的安定 性、水溶性や脂溶性等の物性のコントロールのしやすさなど、脂肪族ホウ酸誘導体と比較して様々 な利点があることが見出されてきた。1 13 年余りに渡り、tavaborole および crisaborole を始めとする、様々な含ホウ素低分子医薬品の研究 開発の現場にいた経験を元に、含ホウ素医薬品が従来のものと比べてどう違うのか、その利点およ び課題について紹介する。

参考文献 1) Baker, S. J., Ding, C. Z., Akama, T., Zhang, Y-K., Hernandez, V. S., Xia, Y., Therapeutic potential of boron-containing compounds. (Review) Future Med. Chem., 1 (7), 1275–1288. (2009) 2) Dembitsky, V. M., Smoum, R., Al-Quntar, A. A., Ali, H. A., Pergament, I., Srebnik, M., Natural occur- rence of boron-containing compounds in plants, algae and microorgamisms. Plant Sci., 163, 931–942. (2002) 3) Navon, A., Ciechanover, A., The 26 S proteasome: From basic mechanism to drug targeting. J. Biol. Chem., 284 (49), 33713–33718. (2009) 4) Jinna, S., Finch, J., Spotlight on tavaborole for the treatment of onychomycosis. Drug Des. Devel. Ther., 9, 6185–6190. (2015) 5) Shirley, M., Ixazomib: First global approval. Drugs, 76 (3), 405–411. (2016) 6) http://www.pfizer.com/news/press-release/press-release-detail/pfizer_receives_fda_approval_for_eu- crisa_crisaborole_a_novel_non_steroidal_topical_ointment_for_mild_to_moderate_atopic_dermati- tis_eczema

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一流誌で目立つ論文撤回:その論文、信じられますか? 長田 裕之 (理研 環境資源科学研究センター) [email protected]

2012 年に Nature 誌に驚くべきコメントが掲載された 1)。Amgen 社と MD Anderson が、がん研究で 一流誌に報告された論文を調査したところ、53 報中 6 報(約 11%)しか再現性が得られなかったと のコメントである。2015 年に発表された Global Biological Standards Institute の調査結果では、米国で 前臨床試験に使われている予算は約 6 兆円であるが、その半分の研究成果が再現できないので、約 3 兆円が無駄になっているとのことである 2)。生物実験では、生物の不均一性や抗体の特異性などが 原因となって再現性が得られない場合も多いようだが、最近は、意図的なデータの改ざん、ねつ造 も目立ってきている 3)。 本講演では、私自身の経験(自分で犯したミス、自分が見つけた他者のミス)を紹介し、どうした らデータ解釈の誤りをなくせるか?再現性の低い論文を出さないようにできるか?を、聴衆とともに 考えたい。

参考文献 1) G. Begley & L. M. Ellis: Nature 483: 531 (2012) 2) L. P. Freedman , I. M. Cockburn & T. S. Simcoe: PLOS Biol 13: e1002165 (2015) 3) F. C. Fang, F. G. Steen & A. Casadevall: Proc. Natl. Acad. Sci. USA 109: 17028-17033 (2012)

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18th International Symposium on the Biology of Actinomycetes (ISBA) 見聞録

去る 2017 年 5 月 23 日から 27 日にかけて 18th International Symposium on the Biology of Actinomycetes (ISBA) が韓国済州島にある ICC で開催された。今回はアメリカ、中国、イギリスを含む 33 カ国から 600 近くの参加者が集まった。日本からは約 70 人が参加した。オープニングセレモニーのあと、ま ず、ノーベル賞受賞者である大村智博士による特別講演が行われた。それに続く最初のプレナリーレ

クチャーはハーバード大学教授 Roberto Kolter 氏が勤め、微生物間相互作用に関する最新の研究を紹 介した。2 日目以降、合計 13 のセッション、5 のワークショプ、7 つのプレナリーレクチャーが行わ れた。2 日目の Genetics and Cell Biology のセッションでは GFP などの蛍光ラベリングを駆使し、染 色体の局在や FtsZ リングの形成に関わる因子の機能解析などの発表が行われ、放線菌に独特の細胞 生物学に関して多くを学ぶことができた。3日目には David Sherman 博士による講演が行われ、電子 顕微鏡を駆使したポリケタイド合成酵素の構造変化のダイナミクスに関する発表が行われた。最終日

のプレナリーレクチャーは Giles van Wezel 氏が務め、電子顕微鏡を駆使して放線菌の細胞内の構造の 解析に関する素晴らし発表を行った。いずれも電子顕微鏡を利用した新規発見であり、高解像度の電 子顕微鏡の利用は今後、放線菌研究の発展にも大きく寄与することを感じさせた。ポスターセション

は 2 回に分かれており、合計 301 の発表があった。そのうち、約 130 演題が生合成に関連するもので あり、放線菌における生合成研究の重要さが伺える。また、植物放線菌相互作用や微生物間の相互作 用に関わる演題も増加傾向にあり、今後これらの分野の発展が期待される。最終日にはポスター賞の

発表があり、合計 12 名がポスター賞を受賞した。日本の参加者からは東京大学、河内護之氏の「Soil Cultivation System for Physiological Analysis of Streptomyces griseus」と産業技術総合研究所、菅野学氏 の「 Plant-Associated Streptomyces Consume Atmospheric H2 Usin a High-Affinity Hydrogenase」、東邦大学、 飯坂洋平氏の「Effective Production of New Rosamicin Derivatives by Engineered Micromonospora rosaria

Mutants with Disruption of a Cytochrome P450 Generated Introduction of the D-mycinose Biosynthetic Genes」 が受賞した。最後に次回の ISBA の開催地がトロントであることが告知され、本大会は閉会した。 (東京大学大学院農学生命科学研究科 勝山 陽平)

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大村智先生の特別講演

ポスター発表の様子

ポスター賞授賞

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9th US-Japan Seminar on Natural Product Biosynthesis 見聞録

五年に一度、日米の天然物の生合成研究者が一堂に集まって研究発表を行う 9th US-Japan Seminar on Natural Product Biosynthesis が 5 月 30 日から 6 月 4 日まで米国カリフォルニア州のレークアローヘ ッドで行われました。今回は日米合わせて 49 名の参加者で皆さんそれぞれ 30 分の発表を 6 日間にわ たって行うという、きわめて内容の濃いセミナーでした。SAJ からは東大の阿部郁郎氏、西山真氏、 葛山智久氏、勝山陽平氏、北大の大利徹氏、筑波大学の小林達彦氏、福井県大の濱野吉十氏、広島大 学の荒川賢治氏、理研の長田裕之氏、そして私とたくさんの方が参加しました。これだけ生合成研究 者ばかりが一堂に集まっての発表を聴くと、生合成研究の現代の潮流が理解できてとても興味深いセ

ミナーでした。 生合成研究は以前より深く、細かい結果が求められていると感じました。分析機器の進歩などを巧 みに組み込んで研究を進めて行くことが求められているようです。たとえば結晶構造なら単なるスナ

ップショットではなく各反応段階ごとに解析したり、クライオ EM なども駆使して、よりダイナミッ クに酵素反応をとらえる感じに進んでいます。また、ポスト生合成研究も重要性を増しており、得ら れた生合成反応から新しい化合物の創製やバイオインフォを用いてより簡単により正確に天然物を

探索する手法なども今後の天然物生合成研究の流れなのではないかと感じました。 標高 1 マイルのアローヘッド湖のほとりに建っている UCLA のカンファレンスセンターが会場で したが、その会場が素晴らしく、まるで軽井沢の別荘のような感じ(行ったことないけど)でした。 皆さんで同じところに泊まって文字通り寝食を共にして勉強しましたので、さしずめ合宿のような感

じでしょうか。 8 時から朝食、9 時から 12 時まで午前のセッション、12 時からランチ、13 時から 18 時までフリー タイム、18 時から 20 時まで夕食、20 時から 23 時まで夜のセッションとなっており、お昼がフリー なので、皆さんテニスをしたり散歩したり、部屋で仕事したりして過ごしていました。食事もレスト

ランでみんなで丸テーブルに座って食べているので、本当に合宿のようです。 次回は 5 年後に日本でということになっています。 (東京大学大学院農学生命科学研究科 尾仲 宏康)

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参加者の集合写真(上)

セミナー会場(上) セミナー参加者そろっての夕食風景(右 上) 会場となった UCLA レークアローヘッ ドカンファレンスセンター(右)

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日本放線菌学会賛助会員

中外製薬(株)鎌倉研究所創薬資源研究部 長瀬産業(株)研究開発センター アステラスファーマテック(株)富山技術センター技術開発部 協和発酵キリン(株)研究本部創薬化学研究所 (公財)微生物化学研究会 微生物化学研究所 第一三共 RD ノバーレ(株)合成化学研究部天然物グループ Meiji Seika フアルマ(株)足柄研究所 日本マイクロバイオファーマ(株)研究開発部 合同酒精(株)酵素医薬品研究所 図書室 味の素株式会社・イノベーション研究所 大鵬薬品工業株式会社 天然物フロンティア研究所 トヨタ紡織株式会社 基礎研究所 富士シリシア チーム未来グループ

著作権について

本誌に掲載された論文、抄録、記事等の著作権は、日本放線菌学会に帰属します。これら著作物の 一部または全部をいかなる形式でもそのまま転載しようとするときは、学会事務局から転載許可を得 て下さい。

日本放線菌学会誌 第 31 巻 1 号 ACTINOMYCETOLOGICA 平成 29 年 7 月 14 日発行

編集兼発行 日本放線菌学会 〒292-0818 千葉県木更津市かずさ鎌足 2-5-8 独立行政法人 製品評価技術基盤機構 バイオテクノロジーセンター(NBRC) 生物資源利用促進課内 TEL 0438-20-5763 FAX 0438-52-2329 E-mail [email protected] 年間購読料 5,000 円(会員無料) http://www.actino.jp/

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日 本 I 放 線 C 菌 学 会 http://www0.nih.go.jp/saj/index-j.html 日本放線菌学会誌 第31巻1号 誌 Published by ACTINOMYCETOLOGICA VOL.31 NO.1, 2017 The Society for Actinomycetes Japan