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A B C D

1990年12月18日第４種郵便物認可 ISSN 0914-5818 2019

VOL. 33 NO. 1

C 2019 T VOL. 33 NO. 1 IN （公開用） O ACTINOMYCETOLOGICA

O L

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. 33, No. 1, 2019

Contents

• Outline of SAJ: Activities and Membership S2 • List of New Scientific Names and Nomenclatural Changes in the Phylum Actinobacteria Validly Published in 2018 S3 • Award Lecture (Dr. Yasuhiro Igarashi) S50 • Publication of Award Lecture (Dr. Yasuhiro Igarashi) S55 • Award Lecture (Dr. Yuki Inahashi) S56 • Publication of Award Lecture (Dr. Yuki Inahashi) S64 • Award Lecture (Dr. Yohei Katsuyama) S65 • Publication of Award Lecture (Dr. Yohei Katsuyama) S72 • 64th Regular Colloquim S73 • 65th Regular Colloquim S74 • The 2019 Annual Meeting of the Society for Actinomycetes Japan S75 • Online access to The Journal of Antibiotics for SAJ members S76

S1 Outline of SAJ: Activities and Membership

The Society for Actinomycetes Japan (SAJ) Annual membership fees are currently 5,000 yen was established in 1955 and authorized as a for active members, 3,000 yen for student mem- scientific organization by Science Council of Japan bers and 20,000 yen or more for supporting mem- in 1985. The Society for Applied Genetics of bers (mainly companies), provided that the fees Actinomycetes, which was established in 1972, may be changed without advance announce- merged in SAJ in 1990. SAJ aims at promoting ment. actinomycete researches as well as social and The current members (April 2018 - March 2020) scientific exchanges between members of the Board of Directors are: Masayuki Hayakawa domestically and internationally. The Activities of (Chairperson; Univ. of Yamanashi), Yasuo SAJ have included annual and regular scientific Ohnishi (Vice Chairperson; Univ. of Tokyo), meetings, workshops and publications of The Atsuko Matsumoto (Secretary General; Kitasato Journal of Antibiotics (the official journal, joint Univ.), Akira Arisawa (MicroBiopharm Japan Co., publication with Japan Antibiotics Research Ltd.), Masayuki Igarashi (Inst. of Microb. Chem. Association), Actinomycetologica (Newsletter) (BIKAKEN)), Susumu Iwamoto (Kyowa Hakko and laboratory manuals. Contributions to Kirin Co., Ltd.), Miyuki Otsuka (Tamagawa International Streptomyces Project (ISP) and Univ.), Hisashi Kawasaki (Univ. of Tokyo), International Symposium on Biology of Masaaki Kizuka (Daiichi Sankyo RD Novare Actinomycetes (ISBA) have also been SAJ's Co., Ltd.), Hideaki Takano (Nihon Univ.), Shunji activities. In addition, SAJ have occasional special Takahashi (RIKEN), Takuji Nakashima (Kitasato projects such as the publication of books related to Univ.), Yoshimitsu Hamano (Fukui Pref. Univ.), actinomycetes: “Atlas of Actinomycetes, 1997”, Hideki Yamamura (Univ. of Yamanashi), Hisayuki “Identification Manual of Actinomycetes, 2001” Komaki (NITE), Masahiro Natsume (Tokyo Univ. and “Digital Atlas of Actinomycetes, 2002” of Agr. & Tech.) and Hideyuki Muramatsu (Inst. (http://atlas.actino.jp/). These activities have of Microb. Chem. (BIKAKEN)) . been planned and organized by the board of The members of the Advisory Board are: directors with association of executive committees Hiroyuki Osada (RIKEN), Keiko Ochiai, Ken- consisting of active members who belong to ichiro Suzuki (Tokyo Univ. Agr.) and Taichi academic and nonacademic organizations. Manome. The SAJ Memberships comprise active members, student members, supporting members and honorary members. Currently (as Copyright: The copyright of the articles of Dec. 20, 2017), SAJ has about 448 active published in Actinomycetologica is transferred members including student members, 21 oversea from the authors to the publisher, The Society for members, 14 honorary members, 3 oversea Actinomycetes Japan, upon acceptance of the honorary members, and 13 supporting members. manuscript. The SAJ members are allowed to join the scientific and social meetings or projects (regular The SAJ Secretariat and specific) of SAJ on a membership basis c/o Institute of Microbial Chemistry (BIKAKEN) and to browse The Journal of Antibiotics from a 3-14-23 Kamiosaki, Shinagawa-ku, link on the SAJ website and will receive each Tokyo 141-0021, JAPAN issue of Actinomycetologica, currently pub- Phone: +81-3-6455-7169 lished in June and December. Actinomycete Fax: +81-3-3441-7589 researchers in foreign countries are welcome to E-mail: [email protected] join SAJ. For application of SAJ membership, please contact the SAJ secretariat (see below).

S2 List of new scientific names and nomenclatural changes in the phylum Actinobacteria validly published in 2018

NEW ORDER

Cryptosporangiales Nouioui et al. 2018, ord. Sporichthyales Nouioui et al. 2018, ord. nov. nov. Type genus: Sporichthya Lechevalier et al. Type genus: Cryptosporangium Tamura et al. 1968. 1998. Reference: Front Microbiol., 2018, 9: 2007; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Microbiol., 2018, 68: 3379–3393]. A member of the class Actinobacteria. A member of the class Actinobacteria.

NEW FAMILY

Actinopolymorphaceae Nouioui et al. 2018, Reference: Front Microbiol., 2018, 9: 2007; fam. nov. Validation List no. 184 [Int J Syst Evol Type genus: Actinopolymorpha Wang et al. Microbiol., 2018, 68: 3379–3393]. 2001. Reference: Front Microbiol., 2018, 9: 2007; Kribbellaceae Nouioui et al. 2018, fam. nov. Validation List no. 184 [Int J Syst Evol Type genus: Kribbella Park et al. 1999 emend. Microbiol., 2018, 68: 3379–3393]. Sohn et al. 2003 emend. Everest et al. 2013. A member of the order Propionibacteriales. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Antricoccaceae Nouioui et al. 2018, fam. nov. Microbiol., 2018, 68: 3379–3393]. Type genus: Antricoccus Lee 2015. A member of the order Propionibacteriales. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Kytococcaceae Nouioui et al. 2018, fam. nov. Microbiol., 2018, 68: 3379–3393]. Type genus: Kytococcus Stackebrandt et al. 1995. Ilumatobacteraceae Asem et al. 2018, fam. Reference: Front Microbiol., 2018, 9: 2007; nov. Validation List no. 184 [Int J Syst Evol Type genus: Ilumatobacter Matsumoto et al. Microbiol., 2018, 68: 3379–3393]. 2009. A member of the order Micrococcales. Reference: Int J Syst Evol Microbiol., 2018, 68: 3593–3599. Lawsonellaceae Nouioui et al. 2018, fam. nov. A member of the order Acidimicrobiales. Type genus: Lawsonella Bell et al. 2016. Reference: Front Microbiol., 2018, 9: 2007; Jatrophihabitantaceae Nouioui et al. 2018, Validation List no. 184 [Int J Syst Evol fam. nov. Microbiol., 2018, 68: 3379–3393]. Type genus: Jatrophihabitans Madhaiyan et al. A member of the order Corynebacteriales. 2013.

S3 Ornithinimicrobiaceae Nouioui et al. 2018, Reference: Front Microbiol., 2018, 9: 2007; fam. nov. Validation List no. 184 [Int J Syst Evol Type genus: Ornithinimicrobium Groth et al. Microbiol., 2018, 68: 3379–3393]. 2001. A member of the order Micrococcales.

NEW GENUS

Aldersonia Nouioui et al. 2018, gen. nov. Type species: Aldersonia kunmingensis (Wang Ellagibacter Beltrán et al. 2018, gen. nov. et al. 2008) Nouioui et al. 2018. Type species: Ellagibacter isourolithinifaciens Reference: Front Microbiol., 2018, 9: 2007; Beltrán et al. 2018. Validation List no. 184 [Int J Syst Evol Reference: Int J Syst Evol Microbiol., 2018, Microbiol., 2018, 68: 3379–3393]. 68: 1707–1712. A member of the family Nocardiaceae. A member of the family Eggerthellaceae.

Boudabousia Nouioui et al. 2018, gen. nov. Embleya Nouioui et al. 2018, gen. nov. Type species: Boudabousia marimammalium Type species: Embleya scabrispora (Ping et al. (Hoyles et al. 2001) Nouioui et al. 2018. 2004) Nouioui et al. 2018. Reference: Front Microbiol., 2018, 9: 2007; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Microbiol., 2018, 68: 3379–3393]. A member of the family Actinomycetaceae. A member of the family Streptomycetaceae.

Bowdeniella corrig. Nouioui et al. 2018, gen. Enteroscipio Danylec et al. 2018, gen. nov. nov. Type species: Enteroscipio rubneri Danylec et Type species: Bowdeniella nasicola corrig. al. 2018. (Hall et al. 2003) Nouioui et al. 2018. Reference: Int J Syst Evol Microbiol., 2018, Reference: Front Microbiol., 2018, 9: 2007; 68: 1533–1540. Validation List no. 184 [Int J Syst Evol A member of the family Eggerthellaceae. Microbiol., 2018, 68: 3379–3393]. A member of the family Actinomycetaceae. Epidermidibacterium Lee et al. 2018, gen. nov. Buchananella Nouioui et al. 2018, gen. nov. Type species: Epidermidibacterium keratini Type species: Buchananella hordeovulneris Lee et al. 2018. (Buchanan et al. 1984) Nouioui et al. 2018. Reference: Int J Syst Evol Microbiol., 2018, Reference: Front Microbiol., 2018, 9: 2007; 68: 745–750]. Validation List no. 184 [Int J Syst Evol A member of the family Sporichthyaceae. Microbiol., 2018, 68: 3379–3393]. A member of the family Actinomycetaceae. Falsarthrobacter Busse and Moo 2018, gen. nov. Desertimonas Asem et al. 2018, gen. nov. Type species: Falsarthrobacter nasiphocae Type species: Desertimonas flava Asem et al. (Collins et al. 2002) Busse and Moo 2018. 2018. Reference: Int J Syst Evol Microbiol., 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: 1361–1364]. 68: 3593–3599. A member of the family Micrococcaceae. A member of the family Ilumatobacteraceae.

S4 Fannyhessea Nouioui et al. 2018, gen. nov. Reference: Front Microbiol., 2018, 9: 2007; Type species: Fanneyhessea vaginae Validation List no. 184 [Int J Syst Evol (Rodriguez Jovita et al. 1999) Nouioui et al. Microbiol., 2018, 68: 3379–3393]. 2018. A member of the family Atopobiaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Libanicoccus Bilen et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Libanicoccus massiliensis A member of the family Atopobiaceae. Bilen et al. 2018. Reference: New Microbes New Infect., 2018, Flavimobilis Nouioui et al. 2018, gen. nov. 21: 63–71; Validation List no. 181 [Int J Syst Type species: Flavimobilis marinus (Huang et Evol Microbiol., 2018, 68: 1411–1417]. al. 2005) Nouioui et al. 2018. A member of the family Coriobacteriaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Longivirga Qu et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Longivirga aurantiaca Qu et al. A member of the family Jonesiaceae. 2018. Reference: Int J Syst Evol Microbiol., 2018, Gleimia Nouioui et al. 2018, gen. nov. 68: 942–946. Type species: Gleimia europaea (Funke et al. A member of the family Sporichthyaceae. 1997) Nouioui et al. 2018. Reference: Front Microbiol., 2018, 9: 2007; Mycobacteroides Gupta et al. 2018, gen. nov. Validation List no. 184 [Int J Syst Evol Type species: Mycobacteroides abscessus Microbiol., 2018, 68: 3379–3393]. (Moore and Frerichs 1953) Gupta et al. A member of the family Actinomycetaceae. 2018. Reference: Front Microbiol., 2018, 9: 67; Jongsikchunia Nouioui et al. 2018, gen. nov. Validation List no. 181 [Int J Syst Evol Type species: Jongsikchunia kroppenstedtii Microbiol., 2018, 68: 1411–1417]. (Kim et al. 2009) Nouioui et al. 2018. A member of the family Mycobacteriaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Mycolicibacillus Gupta et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Mycolicibacillus trivialis A member of the family Gordoniaceae. (Kubica et al. 1970) Gupta et al. 2018. Reference: Front Microbiol., 2018, 9: 67; Klenkia Montero-Calasanz et al. 2018, gen. Validation List no. 181 [Int J Syst Evol nov. Microbiol., 2018, 68: 1411–1417]. Type species: Klenkia marina Montero- A member of the family Mycobacteriaceae. Calasanz et al. 2018. Reference: Front Microbiol., 2017, 8: 2501; Mycolicibacter Gupta et al. 2018, gen. nov. Validation List no. 181 [Int J Syst Evol Type species: Mycolicibacter terraes (Wayne Microbiol., 2018, 68: 1411–1417]. 1966) Gupta et al. 2018. A member of the family Geodermatophilaceae. Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Lancefieldella Nouioui et al. 2018, gen. nov. Microbiol., 2018, 68: 1411–1417]. Type species: Lancefieldella parvula A member of the family Mycobacteriaceae. (Weinberg et al. 1937) Nouioui et al. 2018. Mycolicibacterium Gupta et al. 2018, gen. nov.

S5 Type species: Mycolicibacterium fortuitum (da A member of the family Eggerthellaceae. Costa Cruz 1938) Gupta et al. 2018. Reference: Front Microbiol., 2018, 9: 67; Schaalia Nouioui et al. 2018, gen. nov. Validation List no. 181 [Int J Syst Evol Type species: Schaalia odontolytica (Batty Microbiol., 2018, 68: 1411–1417]. 1958) Nouioui et al. 2018. A member of the family Mycobacteriaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Parolsenella Sakamoto et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Parolsenella catena Sakamoto et A member of the family Actinomycetaceae. al. 2018. Reference: Int J Syst Evol Microbiol., 2018, Thermostaphylospora Wu et al. 2018, gen. 68: 1165–1172. nov. A member of the family Atopobiaceae. Type species: Thermostaphylospora grisealba Wu et al. 2018. Pauljensenia Nouioui et al. 2018, gen. nov. Reference: Int J Syst Evol Microbiol., 2018, Type species: Pauljensenia hongkongensis 68: 602–608. (Woo et al. 2004) Nouioui et al. 2018. A member of the family Streptosporangiaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Winkia Nouioui et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Winkia neuii (Funke et al. 1994) A member of the family Streptosporangiaceae. Nouioui et al. 2018. Reference: Front Microbiol., 2018, 9: 2007; Pedococcus Nouioui et al. 2018, gen. nov. Validation List no. 184 [Int J Syst Evol Type species: Pedococcus dokdonensis (Yoon Microbiol., 2018, 68: 3379–3393]. et al. 2008) Nouioui et al. 2018. A member of the family Actinomycetaceae. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Yinghuangia Nouioui et al. 2018, gen. nov. Microbiol., 2018, 68: 3379–3393]. Type species: Yinghuangia aomiensis (Nagai et A member of the family Intrasporangiaceae. al. 2011) Nouioui et al. 2018. Reference: Front Microbiol., 2018, 9: 2007; Rubneribacter Danylec et al. 2018, gen. nov. Validation List no. 184 [Int J Syst Evol Type species: Rubneribacter badeniensis Microbiol., 2018, 68: 3379–3393]. Danylec et al. 2018. A member of the family Streptomycetaceae. Reference: Int J Syst Evol Microbiol., 2018, 68: 1533–1540.

NEW SPECIES

Acidipropionibacterium virtanenii Deptula et Type strain: strain A251 = CGMCC 4.7421 = al. 2018, sp. nov. JCM 32178. Type strain: strain JS278 = VTT E-113202 = Reference: Int J Syst Evol Microbiol., 2018, 68: DSM 106790. 2325–2330. Reference: Int J Syst Evol Microbiol., 2018, 68: 3175–3183. Actinomadura barringtoniae Rachniyom et al. 2018, sp. nov. Actinocorallia populi Li et al. 2018, sp. nov.

S6 Type strain: strain GKU 128 = TBRC 7225 = Actinorectispora metalli Cao et al. 2018, sp. NBRC113074. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain KC 198 = CCTCC AA 1584–1590. 2015043 = KCTC 39718. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinomadura deserti Cao et al. 2018, sp. nov. 1023–1027. Type strain: strain BMP B8004 = CGMCC 4.7432 = KCTC 39998. Actinotalea solisilvae Yan et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain THG-T121 = KACC 19191 2930–2935. = CGMCC 4.7389. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinomadura rhizosphaerae Malisorn et al. 788–794. 2018, sp. nov. Type strain: strain SDA37 = KCTC 39965 = Aestuariimicrobium soli Chen et al. 2018, sp. NBRC 112909 = TISTR 2523. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain D6 = KCTC 39995 = DSM 3012–3016. 105824. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinomyces tangfeifanii Meng et al. 2018, sp. 3296–3300. nov. Type strain: strain VUL4_3 = CGMCC 4.7369 Amnibacterium endophyticum Li et al. 2018, = DSM 103436. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain 1T4Z-3 = KCTC 39983 = 3701–3706. CGMCC 1.16066. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinomycetospora endophytica Sakdapetsiri 1327–1332. et al. 2018, sp. nov. Type strain: strain A-T 8314 = TBRC 5722 = Amycolatopsis antarctica Wang et al. 2018, NBRC 113235. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain AU-G6 = CGMCC 4.7351 = 3017–3021. NBRC 112404. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinophytocola glycyrrhizae Cao et al. 2018, 2348–2356. sp. nov. Type strain: strain BMP B8152 = KCTC 49002 Amycolatopsis oliviviridis Penkhrue et al. = CGMCC 4.7433. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain SCM_MK2-4 = TBRC 7186 2504–2508. = JCM 32134. Reference: Int J Syst Evol Microbiol., 2018, 68: Actinoplanes sediminis Qu et al. 2018, sp. 1448–1454. nov. Type strain: strain M4I47 = CCTCC AA Amycolatopsis rhizosphaerae Thawai 2018, 2016022 = DSM 100965. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain DH51B-4-3 = TBRC 6029 = 71–75. NBRC 112509. Reference: Int J Syst Evol Microbiol., 2018, 68: 1546–1551.

S7 Reference: Int J Syst Evol Microbiol., 2018, 68: Amycolatopsis silviterrae Jamjan et al. 2018, 575–581. sp. nov. Type strain: strain C12CA1 = TBRC 1456 = Bifidobacterium criceti Lugli et al. 2018, sp. NBRC 111116. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain Ham19E = CCUG 70962 = 1455–1460. LMG 30188. Reference: Syst Appl Microbiol., 2018, 41: Arthrobacter paludis Zhang et al. 2018, sp. 173–183; Validation List no. 182 [Int J Syst nov. Evol Microbiol., 2018, 68: 2130–2133]. Type strain: strain CAU 9143 = KCTC 13958 = CECT 8917. Bifidobacterium imperatoris Lugli et al. 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: sp. nov. 47–51. Type strain: strain Tam1G = CCUG 70961 = LMG 30297. Arthrobacter ruber Liu et al. 2018, sp. nov. Reference: Syst Appl Microbiol., 2018, 41: Type strain: strain MDB1-42 = CGMCC 173–183; Validation List no. 182 [Int J Syst 1.9772 = NBRC 113088. Evol Microbiol., 2018, 68: 2130–2133]. Reference: Int J Syst Evol Microbiol., 2018, 68: 1616–1621. Bifidobacterium italicum Lugli et al. 2018, sp. nov. Arthrobacter silvisoli Yan et al. 2018, sp. nov. Type strain: strain Rab10A = CCUG 70963 = Type strain: strain NEAU-SA1 = DSM 106716 LMG 30187. = CCTCC AB 2017271. Reference: Syst Appl Microbiol., 2018, 41: Reference: Int J Syst Evol Microbiol., 2018, 68: 173–183; Validation List no. 182 [Int J Syst 3892–3896. Evol Microbiol., 2018, 68: 2130–2133].

Bifidobacterium anseris Lugli et al. 2018, sp. Bifidobacterium margollesii Lugli et al. 2018, nov. sp. nov. Type strain: strain Goo31D = CCUG 70960 = Type strain: strain Uis1B = CCUG 70959 = LMG 30189. LMG 30296. Reference: Syst Appl Microbiol., 2018, 41: Reference: Syst Appl Microbiol., 2018, 41: 173–183; Validation List no. 182 [Int J Syst 173–183; Validation List no. 182 [Int J Syst Evol Microbiol., 2018, 68: 2130–2133]. Evol Microbiol., 2018, 68: 2130–2133].

Bifidobacterium callitrichidarum Modesto et Bifidobacterium parmae Lugli et al. 2018, sp. al. 2018, sp. nov. nov. Type strain: strain TRI 5 = DSM 103152 = Type strain: strain Uis4E = CCUG 70964 = JCM 31790. LMG 30295. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Syst Appl Microbiol., 2018, 41: 141–148. 173–183; Validation List no. 182 [Int J Syst Evol Microbiol., 2018, 68: 2130–2133]. Bifidobacterium catulorum Modesto et al. 2018, sp. nov. Bifidobacterium porcinum (Zhu et al. 2003) Type strain: strain MRM 8.19 = DSM 103154 Nouioui et al. 2018, sp. nov. = JCM 31794. Bifidobacterium thermacidophilum subsp. porcinum Zhu et al. 2003 Rank elevation.

S8 Type strain: strain P3-14 = JCM 16945 = LMG Reference: Front Microbiol., 2018, 9: 1809; 21689 = CGMCC 1.3009 = DSM 17755 = Validation List no. 184 [Int J Syst Evol NBRC 106099. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Brevibacterium hankyongi Choi et al. 2018, Microbiol., 2018, 68: 3379–3393]. sp. nov. Type strain: strain BS05 = KACC 18875 = Blastococcus atacamensis Castro et al. 2018, LMG 29562. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain P6 = NCIMB 15090 = 2783–2788. NRRL B-65468. Reference: Int J Syst Evol Microbiol., 2018, 68: Clavibacter capsici (Oh et al. 2016) Nouioui et 2712–2721. al. 2018, sp. nov. Clavibacter michiganensis subsp. capsici Oh et Blastococcus litoris Lee et al. 2018, sp. nov. al. 2016 Rank elevation. Type strain: strain GP-S2-8 = KCCM 43275 = Type strain: strain PF008 = KACC 18448 = JCM 32354 = DSM 106127 = KCTC 49078. LMG 29047. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Front Microbiol., 2018, 9: 2007; 3435–3440. Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Blastococcus xanthinilyticus Hezbri et al. 2018, sp. nov. Clavibacter nebraskensis (Davis et al. 1984) Type strain: strain BMG 862 = DSM 46842 = Nouioui et al. 2018, sp. nov. CECT 8884. Clavibacter michiganensis subsp. nebraskensis Reference: Int J Syst Evol Microbiol., 2018, 68: Davis et al. 1984 Rank elevation. 1177–1183. Type strain: ATCC 27794 = DSM 7483 = CCUG 38894 = CIP 105362 = ICMP 3298 = Brachybacterium avium Tak et al. 2018, sp. JCM 9666 = LMG 3700 = LMG 5627 = nov. LMG 7223 = NCPPB 2581 = VKM Ac- Type strain: strain VR2415 = KCTC 39997 = 1404. JCM 32143. Reference: Front Microbiol., 2018, 9: 2007; Reference: Front Microbiol., 2018, 9: 1809; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393] Microbiol., 2018, 68: 3379–3393]. Clavibacter sepedonicus (Davis et al. 1984) Brachybacterium endophyticum Tuo et al. Nouioui et al. 2018, sp. nov. 2018, sp. nov. Clavibacter michiganensis subsp. sepedonicus Type strain: strain M1HQ-2 = KCTC 49087 = Davis et al. 1984 Rank elevation. CGMCC 1.16391. Type strain: DSM 20744 = JCM 9667 = ATCC Reference: Int J Syst Evol Microbiol., 2018, 68: 33113 = CCUG 23908 = CIP 104844 = 3563–3568. CFBP 2049 = ICMP 2535 = LMG 2889 = NCPPB 2137 = VKM Ac-1405. Brachybacterium vulturis Tak et al. 2018, sp. Reference: Front Microbiol., 2018, 9: 2007; nov. Validation List no. 184 [Int J Syst Evol Type strain: strain VM2412 = KCTC 39996 = Microbiol., 2018, 68: 3379–3393] JCM 32142.

S9 Corynebacterium belfantii Dazas et al. 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: sp. nov. 2530–2537. Type strain: strain FRC0043 = CIP 111412 = DSM 105776. Edaphobacter flagellatus Xia et al. 2018, sp. Reference: Int J Syst Evol Microbiol., 2018, 68: nov. 3826–3831. Type strain: strain HZ411 = GDMCC 1.1193 = LMG 30085. Corynebacterium fournieri corrig. Diop et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 2530–2537. Type strain: strain Marseille-P2948 = CSUR P2948 = DSM 103271. Ellagibacter isourolithinifaciens Beltrán et al. Reference: Antonie van Leeuwenhoek, 2018, 2018, sp. nov. 111: 1165–1174; Validation List no. 184 [Int Type strain: strain CEBAS 4A = DSM 104140 J Syst Evol Microbiol., 2018, 68: 3379– = CCUG 70284. 3393]. Reference: Int J Syst Evol Microbiol., 2018, 68: 1707–1712. Corynebacterium godavarianum Jani et al. 2018, sp. nov. Enteroscipio rubneri Danylec et al. 2018, sp. Type strain: strain PRD07 = MCC 3388 = nov. KCTC 39803 = LMG 29598. Type strain: strain ResAG-96 = DSM 105130 = Reference: Int J Syst Evol Microbiol., 2018, 68: JCM 32273. 241–247. Reference: Int J Syst Evol Microbiol., 2018, 68: 1533–1540. Corynebacterium hadale Wei et al. 2018, sp. nov. Epidermidibacterium keratini Lee et al. 2018, Type strain: strain NBT06-6 = MCCC sp. nov. 1K03347 = DSM 105365. Type strain: strain EPI-7 = KCCM 90264 = Reference: Int J Syst Evol Microbiol., 2018, 68: JCM 31644. 1474–1478. Reference: Int J Syst Evol Microbiol., 2018, 68: 745–750. Cryobacterium aureum Liu et al. 2018, sp. nov. Euzebya rosea Yin et al. 2018, sp. nov. Type strain: strain Hh31 = NBRC 107882 = Type strain: strain DSW09 = DMS 104446 = CGMCC 1.11213. MCCC 1K03290. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 1173–1176. 2900–2905.

Desertimonas flava Asem et al. 2018, sp. nov. Frankia canadensis Normand et al. 2018, sp. Type strain: strain SYSU D60003 = KCTC nov. 39917 = NBRC 112924. Type strain: strain ARgP5 = DSM 45898 = Reference: Int J Syst Evol Microbiol., 2018, 68: CECT 9033. 3593–3599. Reference: Int J Syst Evol Microbiol., 2018, 68: 3001–3011. Edaphobacter bradus Xia et al. 2018, sp. nov. Type strain: strain 4MSH08 = GDMCC 1.1317 Frankia irregularis Nouioui et al. 2018, sp. = KCTC 62475. nov.

S10 Type strain: strain G2 = DSM 45899 = CECT Type strain: strain MH2460 = DSM 103727 = 9038. UTMC 2460 = NCCB 100631. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 2883–2890. 2357–2363.

Frankia saprophytica Nouioui et al. 2018, sp. Glycomyces xiaoerkulensis Wang et al. 2018, nov. sp. nov. Type strain: strain CN3 = DSM 105290 = Type strain: strain TRM 41368 = CCTCC AA CECT 9314. 2017005 = KCTC 39932. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 1090–1095. 2722–2726.

Georgenia deserti Hozzein et al. 2018, sp. nov. Herbidospora soli Niemhom and Thawai 2018, Type strain: strain SYSU D8004 = CGMCC sp. nov. 1.15793 = KCTC 39987. Type strain: strain PS42-9 = BCC 46909 = Reference: Int J Syst Evol Microbiol., 2018, 68: NBRC 108780. 1135–1139. Reference: Int J Syst Evol Microbiol., 2018, 68: 294–298. Glycomyces anabasis Zhang et al. 2018, sp. nov. Jatrophihabitans telluris Lee et al. 2018, sp. Type strain: strain EGI 6500139 = JCM 30088 nov. = KCTC 29495. Type strain: strain N237 = KCTC 39922 = Reference: Int J Syst Evol Microbiol., 2018, 68: NRRL B-65477. 1285–1290. Reference: Int J Syst Evol Microbiol., 2018, 68: 1107–1111. Glycomyces dulcitolivorans Mu et al. 2018, sp. nov. Jishengella zingiberis Thawai et al. 2018, sp. Type strain: strain SJ-25 = CGMCC 4.7414 = nov. DSM 105121. Type strain: strain PLAI 1-1 = TBRC 7644 = Reference: Int J Syst Evol Microbiol., 2018, 68: NBRC 113144. 3034–3039. Reference: Int J Syst Evol Microbiol., 2018, 68: 3345–3350. Glycomyces paridis Fang et al. 2018, sp. nov. Type strain: strain CPCC 204357 = DSM Klenkia marina Montero-Calasanz et al. 2018, 102295 = KCTC 39745. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain YIM M13156 = DSM 45722 1578–1583. = CCTCC AB 2012057. Reference: Front Microbiol., 2017, 8: 2501; Glycomyces rhizosphaerae Li et al. 2018, sp. 3345–3350; Validation List no. 181 [Int J nov. Syst Evol Microbiol., 2018, 68: 1411–1417] Type strain: strain NEAU-C11 = CGMCC 4.7396 = DSM 104646. Kocuria uropygialis Braun et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain 36 = DSM 101740 = LMG 223–227. 29265. Reference: Syst Appl Microbiol., 2018, 41: 38– Glycomyces sediminimaris Mohammadipanah 43; Validation List no. 182 [Int J Syst Evol et al. 2018, sp. nov. Microbiol., 2018, 68: 2130–2133].

S11 Reference: Int J Syst Evol Microbiol., 2018, 68: Kocuria uropygioeca Braun et al. 2018, sp. 3528–3533. nov. Type strain: strain 257 = DSM 101741 = LMG Leucobacter japonicus (Clark and Hodgkin 29266. 2015) Nouioui et al. 2018, sp. nov. Reference: Syst Appl Microbiol., 2018, 41: 38– Leucobacter musarum subsp. japonicus Clark 43; Validation List no. 182 [Int J Syst Evol and Hodgkin 2015 Rank elevation. Microbiol., 2018, 68: 2130–2133]. Type strain: strain CBX130 = DSM 27158 = JCM 31936 = CIP 110719. Kribbella monticola Song et al. 2018, sp. nov. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain NEAU-SW521 = CGMCC Validation List no. 184 [Int J Syst Evol 4.7465 = DSM 105770. Microbiol., 2018, 68: 3379–3393]. Reference: Int J Syst Evol Microbiol., 2018, 68: 3441–3446. Leucobacter triazinivorans Sun et al. 2018, sp. nov. Kribbella podocarpi Curtis et al. 2018, sp. nov. Type strain: strain JW-1 = DSM 105188 = Type strain: strain YPL1 = DSM 29424 = LMG 30083. NRRL B-65063. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Antonie van Leeuwenhoek, 2018, 204–210. 111: 875–882; Validation List no. 183 [Int J Syst Evol Microbiol., 2018, 68: 2707–2709]. Libanicoccus massiliensis Bilen et al. 2018, sp nov. Kribbella qitaiheensis Guo et al. 2018, sp. nov. Type strain: strain Marseille-P3237 = CCUG Type strain: strain NEAU-GQTH2-3 = 71182 = CSUR P3237. CGMCC 4.7215 = JCM 30343. Reference: New Microbes New Infect., 2018, Reference: Antonie van Leeuwenhoek, 2015, 21: 63–71; Validation List no. 181 [Int J Syst 107: 1533–1539; Validation List no. 179 [Int J Evol Microbiol., 2018, 68: 1411–1417]. Syst Evol Microbiol., 2018, 68: 1–2]. Longivirga aurantiaca Qu et al. 2018, sp. nov. Kribbella sindirgiensis Ozdemir-Kocak et al. Type strain: strain X5 = CGMCC 4.7317 = 2018, sp. nov. NBRC 112237. Type strain: strain FSN23 = DSM 27082 = Reference: Int J Syst Evol Microbiol., 2018, 68: KCTC 29220. 942–946. Reference: Arch Microbiol., 2017, 199: 1399– 1407; Validation List no. 180 [Int J Syst Evol Marmoricola silvestris Schumann et al. 2018, Microbiol., 2018, 68: 6 93–694]. sp. nov. Type strain: strain S20-100 = DSM 104694 = Lentzea soli Li et al. 2018, sp. nov. LMG 30008. Type strain: strain NEAU-LZC 7 = CCTCC Reference: Int J Syst Evol Microbiol., 2018, 68: AA 2017027 = JCM 32384. 1313–1318. Reference: Int J Syst Evol Microbiol., 2018, 68: 1496–1501. Microbacterium album Yang et al. 2018, sp. nov. Lentzea terrae Li et al. 2018, sp. nov. Type strain: strain SYSU D8007 = CGMCC Type strain: strain NEAU-LZS 42 = CGMCC 1.15794 = KCTC 39990. 4.7428 = DSM 105696. Reference: Int J Syst Evol Microbiol., 2018, 68: 217–222.

S12 Reference: Int J Syst Evol Microbiol., 2018, 68: Microbacterium deserti Yang et al. 2018, sp. 248–253. nov. Type strain: strain SYSU D8014 = CPCC Micromonospora phytophile Carro et al. 2018, 204619 = KCTC39991. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain SG15 = CECT 9369 = DSM 217–222. 105363. Reference: Int J Syst Evol Microbiol., 2018, 68: Microbacterium halophytorum Li et al. 2018, 248–253. sp. nov. Type strain: strain YJYP 303 = CGMCC Mobiluncus holmesii (Spiegel and Roberts 1.16264 = KCTC 49100. 1984) Nouioui et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Mobiluncus curtisii subsp. holmesii Spiegel 3 928–3934. and Roberts 1984 Rank elevation. Type strain: strain BV376-6 = ATCC 35242 = Microbacterium telephonicum Rahi et al. LMG 7786 = CCUG 17762 = NCTC 11657. 2018, sp. nov. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain S2T63 = MCC 2967 = Validation List no. 184 [Int J Syst Evol KACC 18715 = LMG 29293. Microbiol., 2018, 68: 3379–3393]. Reference: Int J Syst Evol Microbiol., 2018, 68: 1052–1058. Modestobacter lacusdianchii Zhang et al. 2018, sp. nov. Microbispora soli Kittisrisopit et al. 2018, sp. Type strain: strain JXJ CY 19 = CPCC 204352 nov. = KCTC 39600. Type strain: strain RTBAU4-9 = TBRC 7648 = Reference: PLoS One, 2016, 11: e0161069; NBRC 113147. Validation List no. 184 [Int J Syst Evol Reference: Int J Syst Evol Microbiol., 2018, 68: Microbiol., 2018, 68: 3379–3393]. 3863–3868. Mycobacterium decipiens Brown-Elliott et al. Microbispora triticiradicis Han et al. 2018, sp. 2018, sp nov. nov. Type strain: strain TBL 1200985 = ATCC Type strain: strain NEAU-HRDPA2-9 = TSD-117 = DSM 105360. CGMCC 4.7399 = DSM 104649. Reference: Int J Syst Evol Microbiol., 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: 68: 3557–3562. 3600–3605. Mycobacterium komaniense Gcebe et al. Micromonospora globbae Kuncharoen et al. 2018, sp. nov. 2018, sp. nov. Type strain: strain GPK 1020 = CIP 110823 = Type strain: strain WPS1-2 = KCTC 39787 = ATCC BAA-2758. NBRC 112325 = TISTR 2405. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 1526–1532. 1073–1077. Mycobacterium kyogaense Nouioui et al. Micromonospora luteiviridis Carro et al. 2018, 2018, sp. nov. sp. nov. Type strain: NCTC 11659 = CECT 9646 = Type strain: strain SGB14 = CECT 9370 = DSM 107316. DSM 105362.

S13 Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain 78 = CGMCC 4.7461 = 3726–3734. DSM 106424. Reference: Int J Syst Evol Microbiol., 2018, 68: Mycobacterium shigaense Fukano et al. 2018, 3874–3880. sp. nov. Type strain: strain UN-152 = JCM 32072 = Nocardioides pelophilus Yan et al. 2018, sp. DSM 46748. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain THG-T63 = KACC 19192 = 2437–2442. CGMCC 4.7388. Reference: Int J Syst Evol Microbiol., 2018, 68: Mycobacterium syngnathidarum Fogelson et 1942–1948. al. 2018, sp. nov. Type strain: strain 27335 = ATCC TSD-89 = Nonomuraea mangrovi Huang et al. 2018, sp. DSM 105112. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain HA15826 = CGMCC 3696–3700. 4.7425 = DSM 105694. Reference: Int J Syst Evol Microbiol., 2018, 68: Nesterenkonia endophytica Li et al. 2018, sp. 3144–3148. nov. Type strain: strain EGI 60016 = CCTCC AB Parolsenella catena Sakamoto et al. 2018, sp. 2017176 = NBRC 112398. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain 2CBEGH3 = JCM 31932 = 2659–2663. DSM 105194. Reference: Int J Syst Evol Microbiol., 2018, 68: Nocardia rhizosphaerihabitans Ding et al. 1165–1172. 2018, sp. nov. Type strain: strain KLBMP S0039 = KCTC Plantactinospora solaniradicis Li et al. 2018, 39693 = CGMCC 4.7329. sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NEAU-FJL1 = CGMCC 192–197. 4.7284 = DSM 100596. Reference: Antonie van Leeuwenhoek, 2018, Nocardioides allogilvus Zhang et al. 2018, sp. 111: 227–235; Validation List no. 183 [Int J nov. Syst Evol Microbiol., 2018, 68: 2707–2709]. Type strain: strain CFH 30205 = KCTC 49020 = CGMCC 4.7457. Propionicimonas ferrireducens Zhou et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 2485–2490. Type strain: strain Y1A-10 4-9-1 = CCTCC AB 2016249 = KCTC 15566 = LMG 29810. Nocardioides currus Park et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain IB-3 = KACC 19522 = JCM 1914–1918. 32672. Reference: Int J Syst Evol Microbiol., 2018, 68: Pseudonocardia lutea Gao et al. 2018, sp. nov. 2977–2982. Type strain: strain NEAU-G57 = JCM 32387 = CGMCC 4.7397. Nocardioides houyundeii Wang et al. 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: sp. nov. 1992–1997.

S14 Pseudonocardia mangrovi Chanama et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 1749–1756. Type strain: strain SMC 195 = TBRC 7778 = NBRC 113150. Rubneribacter badeniensis Danylec et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 2949–2955. Type strain: strain ResAG-85 = DSM 105129 = JCM 32272. Pseudonocardia soli Thawai 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NW8-21 = BCC 58125 = 1533–1540. NBRC 109519. Reference: Int J Syst Evol Microbiol., 2018, 68: Rubrobacter indicoceani Chen et al. 2018, sp. 1307–1312. nov. Type strain: strain SCSIO 08198 = DSM Pseudopropionibacterium rubrum Saito et al. 105148 = CGMCC 1.16398. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain SK-1 = DSM 100122 = 3487–3493. JCM 31317. Reference: Microbiol Immunol., 2018, 62: Saccharomonospora kobensis (Lacey 1989) 388–394; Validation List no. 183 [Int J Syst Nouioui et al. 2018, sp nov. Evol Microbiol., 2018, 68: 2707–2709]. Saccharopolyspora hirsuta subsp. kobensis (ex Iwazaki et al. 1979) Lacey 1989 Rank Psychromicrobium lacuslunae Kiran et al. elevation 2018, sp. nov. Type strain: DSM 44795 = JCM 9109 = ATCC Type strain: strain IHBB 11108 = MTCC 20501 = NBRC 15151 12460 = MCC 2780 = JCM 31143 = KACC Reference: Front Microbiol., 2018, 9: 2007; 19070. Validation List no. 184 [Int J Syst Evol Reference: Int J Syst Evol Microbiol., 2018, 68: Microbiol., 2018, 68: 3379–3393] 3416–3423. Saccharomonospora piscinae Tseng et al. Rathayibacter oskolensis Dorofeeva et al. 2018, sp. nov. 2018, sp. nov. Type strain: strain 06168H-1 = BCRC 16893 = Type strain: strain DL-329 = VKM Ac-2121 = KCTC 19743. LMG 22542. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 1418–1422. 1442–1447. Saccharopolyspora deserti Yang et al. 2018, Rhodococcus electrodiphilus Ramaprasad et sp. nov. al. 2018, sp. nov. Type strain: strain SYSU D8010 = KCTC Type strain: strain JC435 = KCTC 39856 = 39989 = CPCC 204620. LMG 29881 = MCC 3659. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 860–864. 2644–2649. Saccharopolyspora hattusasensis Veyisoglu et Rhodococcus olei Chaudhary and Kim 2018, al. 2018, sp. nov. sp. nov. Type strain: strain CR3506 = DSM 45715 = Type strain: strain Ktm-20 = KEMB 9005-695 KCTC 29104. = KACC 19390 = JCM 32206.

S15 Reference: Antonie van Leeuwenhoek, 2017, Streptacidiphilus torunensis corrig. Golinska 110: 1719–1727; Validation List no. 180 [Int et al. 2018, sp. nov. J Syst Evol Microbiol., 2018, 68: 693–694]. Type strain: strain NF37 = DSM 102291 = NCIMB 15025. Saccharopolyspora maritima Suksaard et al. Reference: Antonie van Leeuwenhoek, 2016, 2018, sp. nov. 109: 1583–1591; Validation List no. 180 [Int Type strain: strain 3SS5-12 = TBRC 7048 = J Syst Evol Microbiol., 2018, 68: 693–694]. NBRC 112863. Reference: Int J Syst Evol Microbiol., 2018, 68: Streptomyces aqsuensis Wang et al. 2018, sp. 3022–3027. nov. Type strain: strain TRM46399 = KCTC 39610 Sphaerisporangium dianthi Xing et al. 2018, = CCTCC AA 2015009. sp nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NEAU-CY18 = CGMCC 2178–2182. 4.7132 =DSM 46736 = JCM 33407. Reference: Antonie van Leeuwenhoek, 2015, Streptomyces boninensis Také et al. 2018, sp. 107: 9–14; Validation List no. 183 [Int J Syst nov. Evol Microbiol., 2018, 68: 2707–2709]. Type strain: strain K11-0400 = NBRC 113073 = TBRC 7755. Sphaerisporangium rhizosphaerae Mu et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 1795–1799. Type strain: strain NEAU-mq3 = CGMCC 4.7429 = JCM 32389. Streptomyces caeni Huang et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain HA15955 = CGMCC 2860–2865. 4.7426 = DSM 105693. Reference: Int J Syst Evol Microbiol., 2018, 68: Stackebrandtia soli Li et al. 2018, sp. nov. 3080–3083. Type strain: strain AN130378 = DSM 103573 = KCTC 39809. Streptomyces capitiformicae Jiang et al. 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: sp. nov. 1215–1219. Type strain: strain 1H-SSA4 = CGMCC 4.7403 = DSM 104537. Streptacidiphilus monticola Song et al. 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: sp. nov. 118–124. Type strain: strain NEAU-SW11 = CGMCC 4.7427 = DSM 105744. Streptomyces cinnabarigriseus Landwehr et Reference: Int J Syst Evol Microbiol., 2018, 68: al. 2018, sp. nov. 1757–1761. Type strain: strain JS360 = NCCB 100590 = DSM 101724. Streptacidiphilus pinicola Roh et al. 2018, sp. Reference: Int J Syst Evol Microbiol., 2018, 68: nov. 382–393. Type strain: strain MMS16-CNU450 = KCTC 49008 = JCM 32300. Streptomyces davaonensis Landwehr et al. Reference: Int J Syst Evol Microbiol., 2018, 68: 2018, sp. nov. 3149–3155. Type strain: JCM 4913 = DSM 101723. Reference: Int J Syst Evol Microbiol., 2018, 68: 382–393.

S16

Streptomyces dengpaensis Li et al. 2018, sp. Streptomyces salilacus Luo et al. 2018, sp. nov. nov. Type strain: strain XZHG99 = CGMCC 4.7472 Type strain: strain TRM 41337 = CCTCC AA = KCTC 49090. 2015030 = KCTC 39726. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Int J Syst Evol Microbiol., 2018, 68: 3322–3326. 1514–1518.

Streptomyces durbertensis Yu et al. 2018, sp. Streptomyces sediminis Ay et al. 2018, sp. nov. nov. Type strain: strain NEAU-S1GS20 = CCTCC Type strain: strain MKSP12 = DSM 100692 = AA 2017006 = DSM 104538. KCTC 39613. Reference: Int J Syst Evol Microbiol., 2018, 68: Reference: Antonie van Leeuwenhoek, 2018, 3635–3640. 111: 493–500; Validation List no. 182 [Int J Syst Evol Microbiol., 2018, 68: 2130–2133]. Streptomyces fuscigenes Lee and Whang. 2018, sp. nov. Streptomyces swartbergensis le Roes-Hill et Type strain: strain JBL-20 = KACC 18269 = al. 2018, sp. nov. NBRC 110629. Type strain: strain HMC13 = LMG 28849 = Reference: Int J Syst Evol Microbiol., 2018, 68: NRRL B-65294. 1541–1545. Reference: Antonie van Leeuwenhoek, 2018, 111: 589–600; Validation List no. 182 [Int J Streptomyces geranii Li et al. 2018, sp. nov. Syst Evol Microbiol., 2018, 68: 2130–2133]. Type strain: strain A301 = CGMCC 4.7422 = JCM 32177. Streptomyces tritici Zhao et al. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NEAU-A4 = CGMCC 2562–2567. 4.7393 = DSM 104540. Reference: Int J Syst Evol Microbiol., 2018, 68: Streptomyces lichenis Saeng-in et al. 2018, sp. 492–497. nov. Type strain: strain LCR6-01 = KCTC 39908 = Streptomyces triticisoli Tian et al. 2018, sp. TISTR 2500. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NEAU-DSCPA1-4-4 = 3641–3646. CCTCC AA 2017025 = DSM 105118. Reference: Int J Syst Evol Microbiol., 2018, 68: Streptomyces manganisoli Mo et al. 2018, sp. 3327–3332. nov. Type strain: strain MK44 = GDMCC 4.137 = Streptomyces venetus Sujarit et al. 2018, sp. KCTC 39920. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain CMU-AB225 = JCM 31290 1890–1895. = TBRC 2001. Reference: Int J Syst Evol Microbiol., 2018, 68: Streptomyces populi Wang et al. 2018, sp. nov. 3333–3339. Type strain: strain A249 = CGMCC 4.7417 = JCM 3217. Subtercola vilae Villalobos et al. 2018, sp. Reference: Int J Syst Evol Microbiol., 2018, 68: nov. 2568–2573.

S17 Type strain: strain DB165 = DSM 105013 = Reference: Int J Syst Evol Microbiol., 2018, 68: JCM 32044. 602–608. Reference: Antonie van Leeuwenhoek, 2018, 111: 955–963; Validation List no. 184 [Int J Tsukamurella hominis Teng et al. 2018, sp. Syst Evol Microbiol., 2018, 68: 3379–3393]. nov. Type strain: strain HKU65 = JCM 31971 = Tessaracoccus aquimaris Tak et al. 2018, sp. DSM 105036. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain NSG39 = KACC 17540 = 810–818. JCM 19289. Reference: Int J Syst Evol Microbiol., 2018, 68: Tsukamurella ocularis Teng et al. 2018, sp. 1065–1072. nov. Type strain: strain HKU63 = JCM 31969 = Tessaracoccus terricola Chaudhary and Kim DSM 105034. 2018, sp. nov. Reference: Int J Syst Evol Microbiol., 2018, 68: Type strain: strain Brt-A = KEMB 9005-690 = 810–818. KACC 19391 = JCM 32157. Reference: Int J Syst Evol Microbiol., 2018, 68: Virgisporangium myanmarense Yamamura et 529–535. al. 2018, sp. nov. Type strain: strain MM04-1133 = NBRC Thermostaphylospora grisealba Wu et al. 112733 = VTCC 910008. 2018, sp. nov. Reference: J Antibiot., 2017, 70: 995–999; Type strain: strain 3-12X = DSM 46781 = Validation List no. 179 [Int J Syst Evol CGMCC 4.7160. Microbiol., 2018, 68: 1–2].

NEW SUBSPECIES

Adlercreutzia equolifaciens subsp. celatus Reference: Front Microbiol., 2018, 9: 2007; (Minamida et al. 2008) Nouioui et al. 2018, Validation List no. 184 [Int J Syst Evol subsp. nov. Microbiol., 2018, 68: 3379–3393]. Asaccharobacter celatus Minamida et al. 2008 emend. Danylec et al. 2018 Rank reduction. Bifidobacterium catenulatum subsp. Type strain: strain do03 = DSM 18785 = JCM catenulatum (Scardovi and Crociani 1974) 14811 = AHU 1763. Nouioui et al. 2018, subsp. nov. Reference: Front Microbiol., 2018, 9: 2007; Bifidobacterium catenulatum Scardovi and Validation List no. 184 [Int J Syst Evol Crociani 1974 Rank reduction. Microbiol., 2018, 68: 3379–3393]. Type strain: strain B669 = ATCC 27539 = DSM 16992 = DSM 20103 = JCM 1194 = Adlercreutzia equolifaciens subsp. KCTC 3221. equolifaciens (Maruo et al. 2008) Nouioui et Reference: Front Microbiol., 2018, 9: 2007; al. 2018, subsp. nov. Validation List no. 184 [Int J Syst Evol Adlercreutzia equolifaciens Maruo et al. 2008 Microbiol., 2018, 68: 3379–3393]. Rank reduction. Type strain: strain FJC-B9 = CCUG 54925 = Bifidobacterium catenulatum subsp. DSM 19450 = JCM 14793. kashiwanohense (Morita et al. 2011) Nouioui et al. 2018, subsp. nov.

S18 Bifidobacterium kashiwanohense Morita et al. Type strain: ATCC 6919 = CCUG 1794 = CIP 2011 Rank reduction. 53.117 = DSM 1897 = JCM 6425 = LMG Type strain: strain HM2-2 = DSM 21854 = 16711 = NCTC 737 = NRRL B-4224 = JCM 15439. VKM Ac-1450 = BCRC 10723 = CECT Reference: Front Microbiol., 2018, 9: 2007; 5684 = CGMCC 1.5003 = KCTC 3314 = Validation List no. 184 [Int J Syst Evol KCTC 5008 = LMG 3591 = NBRC 107605 Microbiol., 2018, 68: 3379–3393]. = NCTC 737= NRRL B-4224 = VKM Ac- 1450 = VPI 0389. Bifidobacterium pullorum subsp. gallinarum Reference: Front Microbiol., 2018, 9: 2007; (Watabe et al. 1983) Nouioui et al. 2018, Validation List no. 184 [Int J Syst Evol subsp. nov. Microbiol., 2018, 68: 3379–3393]. Bifidobacterium gallinarum Watabe et al. 1983 Rank reduction. Dietzia kunjamensis subsp. kunjamensis Type strain: strain Ch206-5 = DSM 20670 = (Mayilraj et al. 2006) Nouioui et al. 2018, JCM 6291 = AS 1.2283 = ATCC 33777 = subsp. nov. BCRC 14679 = CCUG 34990 = LMG [Dietzia kunjamensis Mayilraj et al. 2006] 11586. Rank reduction. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain K30-10 = DSM 44907 = Validation List no. 184 [Int J Syst Evol JCM 13325 = MTCC 7007. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Bifidobacterium pullorum subsp. pullorum Microbiol., 2018, 68: 3379–3393]. (Trovatelli et al. 1974) Nouioui et al. 2018, subsp. nov. Dietzia kunjamensiss subsp. schimae Type strain: ATCC 27685 = BCRC 14678 = (Minamida et al. 2008) Nouioui et al. 2018, CCUG 35235 = CGMCC 1.2240 = CIP subsp. nov. 107006 = DSM 20433 = JCM 1214 = LMG Dietzia schimae Li et al. 2008 Rank reduction. 11572. Type strain: DSM 45139 = JCM 16003 = Reference: Front Microbiol., 2018, 9: 2007; CCTCC AA 207015. Validation List no. 184 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 2007; Microbiol., 2018, 68: 3379–3393]. Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Bifidobacterium pullorum subsp. saeculare (Biavati et al. 1992) Nouioui et al. 2018, Lentzea albidocapillata subsp. albidocapillata subsp. nov. (Yassin et al. 1995) Nouioui et al. 2018, Bifidobacterium saeculare Biavati et al. 1992 subsp. nov. Rank reduction. [Lentzea albidocapillata Yassin et al. 1995] Type strain: strain RA161 = DSM 6531 = JCM Rank reduction. 18223 = AS 1.2278 = ATCC 49392 = Type strain: ATCC 51859 = CCUG 48294 = CCUG 34977 = LMG 14934. CIP 104842 = DSM 44073 = IMMIB D-958 Reference: Front Microbiol., 2018, 9: 2007; = IMSNU 21253 = JCM 9732 = NBRC Validation List no. 184 [Int J Syst Evol 100372 = NRRL B-24057. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Cutibacterium acnes subsp. acnes (Gilchrist Microbiol., 2018, 68: 3379–3393]. 1900) Nouioui et al. 2018, subsp. nov.

S19 Lentzea albidocapillata subsp. violacea (Lee Nocardia salmonicida subsp. cummidelens et al. 2000) Nouioui et al. 2018, subsp. nov. (Maldonado et al. 2001) Nouioui et al. 2018, Saccharothrix violacea Lee et al. 2000 Rank subsp. nov. reduction Nocardia cummidelens Maldonado et al. 2001 Type strain: strain LM 036 = DSM 44796 = Rank reduction JCM 10975. Type strain: strain R89 = DSM 44490 = JCM Reference: Front Microbiol., 2018, 9: 2007; 11439 = CCUG 46295 = CIP 107225 = IFM Validation List no. 184 [Int J Syst Evol 10176 = NBRC 100378 = NCIMB 13758. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Mycobacterium chelonae subsp. gwanakae Microbiol., 2018, 68: 3379–3393]. Kim et al. 2018, subsp. nov. Type strain: strain MOTT36W = KCTC 29127 Nocardia salmonicida subsp. salmonicida = JCM 32454. (Isik et al. 1999) Nouioui et al. 2018, subsp. Reference: Int J Syst Evol Microbiol., 2018, nov. 68: 3772–3780. [Nocardia salmonicida (ex Rucker 1969) Isik et al. 1999] Rank reduction. Mycobacterium intracellulare subsp. Type strain: ATCC 27463 = CBS 694.72 = CIP chimaera (Tortoli et al. 2004) Nouioui et al. 104517 = DSM 40472 = NBRC 13393 = ISP 2018, subsp. nov. 5472 = JCM 4826 = NRRL B-2778 = NRRL Mycobacterium chimaera Tortoli et al. 2004 B-12385 = RIA 1354. Rank reduction. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain FI-01069 = DSM 44623 = Validation List no. 184 [Int J Syst Evol JCM 14737 = CCUG 50989 = CIP 107892. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Saccharomonospora iraqiensis subsp. Microbiol., 2018, 68: 3379–3393]. paurometabolica (Li et al. 2003) Nouioui et al. 2018, subsp. nov. Mycobacterium intracellulare subsp. Saccharomonospora paurometabolica Li et al. intracellulare Castejon et al. 2018, subsp. 2003 Rank reduction. nov. Type strain: DSM 44619 = JCM 13241 = Type strain: strain 3600 (TMC1406) = ATCC BCRC 16315 = CCTCC AA 001018. 13950 = CCUG 28005 = CIP 104243 = Reference: Front Microbiol., 2018, 9: 2007; DSM43223 = JCM 6384 = NCTC 13025. Validation List no. 184 [Int J Syst Evol Reference: Int J Syst Evol Microbiol., 2018, Microbiol., 2018, 68: 3379–3393]. 68: 1998–2005. Winkia neuii subsp. neuii (Funke et al. 1994) Mycobacterium intracellulare subsp. Nouioui et al. 2018, subsp. nov. yongonense Castejon et al. 2018, subsp. Rank reduction. nov. Type strain: strain 97/90 = CCUG 32252 = Type strain: strain 05-1390 = DSM 45126 = DSM 8576 = ATCC 51847 = CIP 104015. KCTC 19555. Reference: Front Microbiol., 2018, 9: 2007; Reference: Int J Syst Evol Microbiol., 2018, Validation List no. 184 [Int J Syst Evol 68: 1998–2005. Microbiol., 2018, 68: 3379–3393].

S20 NEW COMBINATION

Actinokineospora alba (Yuan et al. 2010) Type strain: strain Mt1B8 = CCUG 54980 = Nouioui et al. 2018, comb. nov. DSM 19490. Basonym: Alloactinosynnema album Yuan et Reference: Front Microbiol., 2018, 9: 2007; al. 2010. Validation List no. 184 [Int J Syst Evol Type strain: strain 03-9939 = CCM 7461 = Microbiol., 2018, 68: 3379–3393]. DSM 45114 =KCTC 19294 = NBRC 106985. Adlercreutzia muris (Lagkouvardos et al. Reference: Front Microbiol., 2018, 9: 2007; 2016) Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Enterorhabdus muris Lagkouvardos Microbiol., 2018, 68: 3379–3393]. et al. 2016. Type strain: strain WCA-131-CoC-2 = DSM Actinokineospora iranica (Nikou et al. 2014) 29508 = KCTC 15543. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Alloactinosynnema iranicum Nikou Validation List no. 184 [Int J Syst Evol et al. 2014. Microbiol., 2018, 68: 3379–3393]. Type strain: strain Chem10 = CECT 8209 = IBRC-M 10403. Aldersonia kunmingensis (Wang et al. 2008) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Rhodococcus kunmingensis Wang et Microbiol., 2018, 68: 3379–3393]. al. 2008. Type strain: DSM 45001 = JCM 15626 = Adlercreutzia caecicola (Clavel et al. 2013) KCTC 19149. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Parvibacter caecicola Clavel et al. Validation List no. 184 [Int J Syst Evol 2013. Microbiol., 2018, 68: 3379–3393]. Type strain: strain NR06 = CCUG 57646 = DSM 22242. Amycolatopsis arida (Mao et al. 2011) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Yuhushiella deserti Mao et al. 2011. Microbiol., 2018, 68: 3379–3393]. Type strain: strain RA45 = DSM 45648 = JCM 16584. Adlercreutzia caecimuris (Clavel et al. 2010) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Enterorhabdus caecimuris Clavel et Microbiol., 2018, 68: 3379–3393]. al. 2010. Type strain: strain B7 = CCUG 56815 = DSM Boudabousia marimammalium (Hoyles et al. 21839. 2001) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces marimammalium Validation List no. 184 [Int J Syst Evol Hoyles et al. 2001. Microbiol., 2018, 68: 3379–3393]. Type strain: strain M1749/98/1 = CCUG 41710 = DSM 15383 = CIP 106509. Adlercreutzia mucosicola (Clavel et al. 2009) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Enterorhabdus mucosicola Clavel et Microbiol., 2018, 68: 3379–3393]. al. 2009.

S21 Bowdeniella nasicola corrig. (Hall et al. 2003) Corynebacterium otitidis (Funke et al. 1994) Nouioui et al. 2018, comb. nov. Baek et al. 2018, comb. nov. Basonym: Actinomyces nasicola Hall et al. Basonym: Turicella otitidis Funke et al. 1994. 2003. Type strain: strain 234/92 = DSM 8821 = JCM Type strain: strain R2014 = CCUG 46092 = 12146. CIP 107668. Reference: Front Microbiol., 2018, 9: 834; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 183 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 2707–2709]. Microbiol., 2018, 68: 3379–3393]. Cutibacterium acnes subsp. defendens Buchananella hordeovulneris (Buchanan et (McDowell et al. 2016) Nouioui et al. 2018, al. 1984) Nouioui et al. 2018, comb. nov. comb. nov. Basonym: Actinomyces hordeovulneris Basonym: Propionibacterium acnes subsp. Buchanan et al. 1984. defendens McDowell et al. 2016. Type strain: ATCC 35275 = DSM 20732 = Type strain: CCUG 6369 = JCM 6473 = ATCC CCUG 32937 = CIP 103149 = UCD 81-332- 11828. 9. Reference: Front Microbiol., 2018, 9: 2007; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Microbiol., 2018, 68: 3379–3393]. Cutibacterium namnetense (Aubin et al. 2016) Clavibacter insidiosus (McCulloch 1925) Li et Nouioui et al. 2018, comb. nov. al. 2018, comb. nov. Basonym: Propionibacterium namnetense Basonym: Corynebacterium insidiosum Aubin et al. 2016. (McCulloch 1925) Jensen 1934, Type strain: strain NTS 31307302 = DSM Corynebacterium michiganense subsp. 29427 = CCUG 66358. insidiosum (McCulloch 1925) Carlson and Reference: Front Microbiol., 2018, 9: 2007; Vidaver 1982, Clavibacter michiganensis Validation List no. 184 [Int J Syst Evol subsp insidiosus (McCulloch 1925) Davis et Microbiol., 2018, 68: 3379–3393]. al. 1984. Type strain: ATCC 10253 = LMG 3663 = Demequina gelatinilytica (Hamada et al. 2015) NCPPB 1109. Nouioui et al. 2018, comb. nov. Reference: Int J Syst Evol Microbiol., 2018, Basonym: Lysinimicrobium gelatinilyticum 68: 234–240 Hamada et al. 2015. Type strain: strain HI12-44 = DSM 28149 = Clavibacter tessellarius (Carlson and Vidaver NBRC 109390. 1982) Li et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Corynebacterium michiganense Validation List no. 184 [Int J Syst Evol subsp. tessellarius Carlson and Vidaver Microbiol., 2018, 68: 3379–3393]. 1982, Clavibacter michiganensis subsp. tessellarius corrig. (Carlson and Vidaver Demequina iriomotensis (Hamada et al. 2015) 1982) Davis et al. 1984. Nouioui et al. 2018, comb. nov. Type strain: ATCC 33566 = NCPPB 3664 = Basonym: Lysinimicrobium iriomotense LMG 7294. Hamada et al. 2015. Reference: Int J Syst Evol Microbiol., 2018, Type strain: strain HI12-143 = DSM 28146 = 68: 234–240 NBRC 109399. Reference: Front Microbiol., 2018, 9: 2007;

S22 Validation List no. 184 [Int J Syst Evol Type strain: strain HI12-128 = DSM 28145 = Microbiol., 2018, 68: 3379–3393]. NBRC 109396. Reference: Front Microbiol., 2018, 9: 2007; Demequina mangrovi (Hamada et al. 2012) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Lysinimicrobium mangrovi Hamada et al. 2012. Embleya scabrispora (Ping et al. 2004) Type strain: strain HI08-69 = DSM 24868 = Nouioui et al. 2018, comb. nov. NBRC 105856. Basonym: Streptomyces scabrisporus Ping et Reference: Front Microbiol., 2018, 9: 2007; al. 2004. Validation List no. 184 [Int J Syst Evol Type strain: strain KM-4927 = DSM 41855 = Microbiol., 2018, 68: 3379–3393]. JCM 11712 = NBRC 100760 = NRRL B- 24202. Demequina pelophila (Hamada et al. 2015) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Lysinimicrobium pelophilum Microbiol., 2018, 68: 3379–3393]. Hamada et al. 2015. Type strain: strain HI12-111 = DSM 28148 = Falsarthrobacter nasiphocae (Collins et al. NBRC 109393. 2002) Busse and Moo et al. 2018, comb. Reference: Front Microbiol., 2018, 9: 2007; nov. Validation List no. 184 [Int J Syst Evol Basonym: Arthrobacter nasiphocae Collins et Microbiol., 2018, 68: 3379–3393]. al. 2002. Type strain: strain M597/99/10 = CCUG 42953 Demequina rhizosphaerae (Hamada et al. = CIP 107054 = DSM 13988 = JCM 11677. 2015) Nouioui et al. 2018, comb. nov. Reference: Int J Syst Evol Microbiol., 2018, Basonym: Lysinimicrobium rhizosphaerae 68: 1361-1364. Hamada et al. 2015. Type strain: strain HI12-135 = DSM 28152 = Fannyhessea vaginae (Rodríguez-Jovita et al. NBRC 109397. 1999) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Atopobium vaginae Rodriguez- Validation List no. 184 [Int J Syst Evol Jovita et al. 1999. Microbiol., 2018, 68: 3379–3393]. Type strain: CCUG 38953 = CIP 106431 = ATCC BAA-55. Demequina soli (Hamada et al. 2015) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Lysinimicrobium soli Hamada et al. Microbiol., 2018, 68: 3379–3393]. 2015. Type strain: strain HI12-122 = DSM 28151 = Flavimobilis marinus (Huang et al. 2005) NBRC 109394. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Sanguibacter marinus Huang et al. Validation List no. 184 [Int J Syst Evol 2005 emend. Pikuta et al. 2017. Microbiol., 2018, 68: 3379–3393]. Type strain: strain 1-19 = DSM 19083 = JCM 12547 = CGMCC 1.3457. Demequina subtropica (Hamada et al. 2015) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Lysinimicrobium subtropicum Microbiol., 2018, 68: 3379–3393]. Hamada et al. 2015.

S23 Flavimobilis soli (Kim et al. 2008) Nouioui et Reference: Front Microbiol., 2018, 9: 2007; al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Sanguibacter soli Kim et al. 2008 Microbiol., 2018, 68: 3379–3393]. emend. Pikuta et al. 2017. Type strain: strain DCY22 = JCM 14841 = Gulosibacter faecalis (Lin et al. 2004) Nouioui KCTC 13155. et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Zimmermannella faecalis Lin et al. Validation List no. 184 [Int J Syst Evol 2004. Microbiol., 2018, 68: 3379–3393]. Type strain: CCUG 37873 = JCM 12866 = ATCC 13722 = NBRC 15706 = TISTR Gleimia coleocanis (Hoyles et al. 2002) 1514. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces coleocanis Hoyles et Validation List no. 184 [Int J Syst Evol al. 2002. Microbiol., 2018, 68: 3379–3393]. Type strain: strain M343-98/2 = CIP 106873 = DSM 15436 = CCUG 41708. Haloechinothrix halophila (Tang et al. 2010) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Amycolatopsis halophila Tang et al. Microbiol., 2018, 68: 3379–3393]. 2010. Type strain: DSM 45216 = JCM 18118 = Gleimia europaea (Funke et al. 1997) Nouioui KCTC 19403. et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces europaeus Funke et al. Validation List no. 184 [Int J Syst Evol 1997. Microbiol., 2018, 68: 3379–3393]. Type strain: ATCC 700353 = LMG 18454 = CCUG 32789 A = CIP 105308. Haloechinothrix salitolerans (Guan et al. Reference: Front Microbiol., 2018, 9: 2007; 2012) Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Amycolatopsis salitolerans Guan et Microbiol., 2018, 68: 3379–3393]. al. 2012. Type strain: strain TRM F103 = DSM 45783 = Gleimia hominis (Funke et al. 2010) Nouioui JCM 15899 = CCTCC AB 208326. et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces hominis Funke et al. Validation List no. 184 [Int J Syst Evol 2010. Microbiol., 2018, 68: 3379–3393]. Type strain: strain 1094 = strain 7894GR = CCUG 57540 = DSM 22168. Intrasporangium flavum (Azman et al. 2016) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Monashia flava Azman et al. 2016. Microbiol., 2018, 68: 3379–3393]. Type strain: strain MUSC 78 = DSM 29621 = NBRC 110749 = MCCC 1K00454. Gulosibacter bifidus (Lin et al. 2004) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Zimmermannella bifida Lin et al. Microbiol., 2018, 68: 3379–3393]. 2004. Type strain: DSM 17450 = JCM 12920 = Jongsikchunia kroppenstedtii (Kim et al. CCUG 50209 = NBRC 103089 = TISTR 2009) Nouioui et al. 2018, comb. nov. 1511. Basonym: Gordonia kroppenstedtii Kim et al.

S24 2009. Type strain: strain 3-wff-81 = DSM 45962 = Type strain: strain NP8-5 = DSM 45133 = JCM NBRC 109416 = CGMCC 1.12303. 16948 = KCTC 19360. Reference: Front Microbiol., 2018, 9: 2501; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 181 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417]. Microbiol., 2018, 68: 3379–3393]. Klenkia terrae (Jin et al. 2013) Montero- Kitasatospora indigofera (Shinobu and Calasanz et al. 2018, comb. nov. Kawato 1960) Nouioui et al. 2018, comb. Basonym: Geodermatophilus terrae Jin et al. nov. 2013. Basonym: Streptomyces indigoferus Shinobu Type strain: strain PB261 = JCM 17786 = and Kawato 1960. DSM 45844 = KCTC 19881. Type strain: DSM 40124 = JCM 4646 = ATCC Reference: Front Microbiol., 2018, 9: 2501; 23924 = BCRC 13773 = CBS 908.68 = Validation List no. 181 [Int J Syst Evol NBRC 12878 = NCIMB 9718 = NRRL B- Microbiol., 2018, 68: 1411–1417]. 3301 = RIA 1127. Reference: Front Microbiol., 2018, 9: 2007; Knoellia remsis (Osman et al. 2007) Nouioui Validation List no. 184 [Int J Syst Evol et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Tetrasphaera remsis Osman et al. 2007. Kitasatospora xanthocidica (Asahi et al. 1966) Type strain: strain 3-M5-R-4 = CIP 109413 = Nouioui et al. 2018, comb. nov. JCM 15662 = ATCC BAA-1496. Basonym: Streptomyces xanthocidicus Asahi et Reference: Front Microbiol., 2018, 9: 2007; al. 1966. Validation List no. 184 [Int J Syst Evol Type strain: DSM 40575 = JCM 4243 = ATCC Microbiol., 2018, 68: 3379–3393]. 27480 = BCRC 11874 = CBS 770.72 = CGMCC 4.1424 = ISP 5575 = JCM 4862 = Lentzea aerocolonigenes (Labeda et al. 1986) NBRC 13469 = NRRL B-12504 = NRRL Nouioui et al. 2018, comb. nov. ISP-5575 = VKM Ac-872. Basonym: Saccharothrix aerocolonigenes (ex Reference: Front Microbiol., 2018, 9: 2007; Shinobu and Kawato 1960) Labeda 1986. Validation List no. 184 [Int J Syst Evol Type strain: strain Shinobu 701 = DSM 40034 Microbiol., 2018, 68: 3379–3393]. = JCM 4150 = ATCC 23870 = BCRC 13661 = CIP 107109 = ISP 5034 = JCM 4614 = Klenkia soli (Jin et al. 2013) Montero- NBRC 13195 = NRRL B-3298 = NRRL Calasanz et al. 2018, comb. nov. ISP-5034 = VKM Ac-1081. Basonym: Geodermatophilus soli Jin et al. Reference: Front Microbiol., 2018, 9: 2007; 2013. Validation List no. 184 [Int J Syst Evol Type strain: strain PB34 = DSM 45843 = JCM Microbiol., 2018, 68: 3379–3393]. 17785 = KCTC 19880. Reference: Front Microbiol., 2018, 9: 2501; Lentzea atacamensis (Okoro et al. 2010) Validation List no. 181 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417]. Basonym: Lechevalieria atacamensis Okoro et al. 2010. Klenkia taihuensis (Qu et al. 2014) Montero- Type strain: strain C61 = DSM 45479 = JCM Calasanz et al. 2018, comb. nov. 17492 = CGMCC 4.5536 and NRRL B- Basonym: Geodermatophilus taihensis Qu et 24706. al. 2014. Reference: Front Microbiol., 2018, 9: 2007;

S25 Validation List no. 184 [Int J Syst Evol Lentzea roselyniae (Okoro et al. 2010) Microbiol., 2018, 68: 3379–3393]. Nouioui et al. 2018, comb. nov. Basonym: Lechevalieria roselyniae Okoro et Lentzea deserti (Okoro et al. 2010) Nouioui et al. 2010. al. 2018, comb. nov. Type strain: strain C81 = DSM 45481 = JCM Basonym: Lechevalieria deserti Okoro et al. 17494 = CGMCC 4.5537 = NRRL B-24708. 2010. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain C68 = DSM 45480 = JCM Validation List no. 184 [Int J Syst Evol 17493 = CGMCC 4.5535 and NRRL B- Microbiol., 2018, 68: 3379–3393]. 24707. Reference: Front Microbiol., 2018, 9: 2007; Lentzea xinjiangensis (Wang et al. 2007) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Lechevalieria xinjiangensis Wang et al. 2007. Lentzea flava (Gauze et al. 1974) Nouioui et Type strain: strain R24 = DSM 45081 = JCM al. 2018, comb. nov. 15473 =CGMCC 4.3525 = NBRC 106319. Basonym: Actinomadura flava Gauze et al. Reference: Front Microbiol., 2018, 9: 2007; 1974. Validation List no. 184 [Int J Syst Evol Type strain: DSM 43885 = JCM 3296 =ATCC Microbiol., 2018, 68: 3379–3393]. 29533 = BCRC 13328 = CIP 107110 = NBRC 14521 = NCIB 11447 = NRRL B- Microlunatus aerolatus (Kim et al. 2016) 16131 = VKM Ac-906. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Friedmanniella aerolata Kim et al. Validation List no. 184 [Int J Syst Evol 2016. Microbiol., 2018, 68: 3379–3393]. Type strain: strain 7515T-26 = DSM 27139 = KACC 17306 = NBRC 109600. Lentzea fradiae (Zhang et al. 2007) Nouioui et Reference: Front Microbiol., 2018, 9: 2007; al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Lechevalieria fradiae Zhang et al. Microbiol., 2018, 68: 3379–3393]. 2007. Type strain: strain Z6 = DSM 45099 = JCM Microlunatus antarcticus (Schumann et al. 14205 = CGMCC 4.3506 = NBRC 106318. 1997) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Friedmanniella antarctica Validation List no. 184 [Int J Syst Evol Schumann et al. 1997. Microbiol., 2018, 68: 3379–3393]. Type strain: strain AA-1042 = DSM 11053 = KACC 14501. Lentzea nigeriaca (Camas et al. 2013) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Lechevalieria nigeriaca Camas et al. Microbiol., 2018, 68: 3379–3393]. 2013. Type strain: strain NJ2035 = DSM 45680 = Microlunatus capsulatus (Maszenan et al. JCM 31207 = KCTC 29057 = NRRL B- 1999) Nouioui et al. 2018, comb. nov. 24881. Basonym: Friedmanniella capsulata Maszenan Reference: Front Microbiol., 2018, 9: 2007; et al. 1999. Validation List no. 184 [Int J Syst Evol Type strain: strain Ben 108 = JCM 13522 = Microbiol., 2018, 68: 3379–3393]. KACC 14261 = ACM 5120. Reference: Front Microbiol., 2018, 9: 2007;

S26 Validation List no. 184 [Int J Syst Evol Type strain: strain FB2 = DSM 21743 = KACC Microbiol., 2018, 68: 3379–3393]. 15056 = NBRC 104965. Reference: Front Microbiol., 2018, 9: 2007; Microlunatus flavus (Zhang et al. 2013) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Friedmanniella flava Zhang et al. 2013. Microlunatus spumicola corrig. (Maszenan et Type strain: strain W6 = JCM 17701 = KACC al. 1999) Nouioui et al. 2018, comb. nov. 17380 = CGMCC 4.6856. Basonym: Friedmanniella spumicola Reference: Front Microbiol., 2018, 9: 2007; Maszenan et al. 1999. Validation List no. 184 [Int J Syst Evol Type strain: strain Ben 107 = JCM 16540 = Microbiol., 2018, 68: 3379–3393]. KACC 14247 = ACM 5121. Reference: Front Microbiol., 2018, 9: 2007; Microlunatus lacustris (Lawson et al. 2000) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Friedmanniella lacustris Lawson et al. 2000. Micromonospora andamanensis (Supong et Type strain: strain EL-17a = ATCC BAA-165 = al. 2013) Nouioui et al. 2018, comb. nov. KACC 15021 = DSM 11465. Basonym: Verrucosispora andamanensis Reference: Front Microbiol., 2018, 9: 2007; Supong et al. 2013. Validation List no. 184 [Int J Syst Evol Type strain: strain SP03-05 = DSM 46721 = Microbiol., 2018, 68: 3379–3393]. NBRC 109075 = BCC 45620. Reference: Front Microbiol., 2018, 9: 2007; Microlunatus lucidus (Iwai et al. 2010) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Friedmanniella lucida Iwai et al. 2010. Micromonospora fiedleri (Goodfellow et al. Type strain: strain FA2 = DSM 21742 = KACC 2013) Nouioui et al. 2018, comb. nov. 15053 = NBRC 104964. Basonym: Verrucosispora fiedleri Goodfellow Reference: Front Microbiol., 2018, 9: 2007; et al. 2013. Validation List no. 184 [Int J Syst Evol Type strain: strain MG-37 = DSM 46741 = Microbiol., 2018, 68: 3379–3393]. KACC 18210 = NCIMB 14794 = NRRL B- 24892. Microlunatus okinawensis (Iwai et al. 2010) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Friedmanniella okinawensis Iwai et Microbiol., 2018, 68: 3379–3393]. al. 2010. Type strain: strain FB1 = DSM 21744 = KACC Micromonospora gifhornensis (Rheims et al. 15055 = NBRC 104966. 1998) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Verrucosispora gifhornensis Rheims Validation List no. 184 [Int J Syst Evol et al. 1998. Microbiol., 2018, 68: 3379–3393]. Type strain: strain HR1-2 = DSM 44337 = KACC 20946 = JCM 10457 = NBRC 16317. Microlunatus sagamiharensis (Iwai et al. Reference: Front Microbiol., 2018, 9: 2007; 2010) Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Friedmanniella sagamiharensis Microbiol., 2018, 68: 3379–3393]. corrig. Iwai et al. 20.

S27 Micromonospora lutea (Liao et al. 2009) 1992 emend. Tortoli et al. 2016. Nouioui et al. 2018, comb. nov. Type strain: strain Hauduroy L948 = ATCC Basonym: Verrucosispora lutea Liao et al. 19977 = DSM 44196 = CIP 104536 = 2009. CCUG 20993 = JCM 13569 = NCTC 13031. Type strain: DSM 45424 = JCM 16959 = BCC Reference: Front Microbiol., 2018, 9: 67; 49507 = CCTCC AA207012 = KCTC 19195 Validation List no. 181 [Int J Syst Evol = NBRC 106530. Microbiol., 2018, 68: 1411–1417]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Mycobacteroides abscessus subsp. abscessus Microbiol., 2018, 68: 3379–3393]. (Moore et al. 1953) Gupta et al. 2018, comb. nov. Micromonospora maris (Goodfellow et al. Basonym: Mycobacterium abscessus (Moore 2012) Nouioui et al. 2018, comb. nov. and Frerichs 1953) Kusunoki and Ezaki Basonym: Verrucosispora maris Goodfellow et 1992 emend. Tortoli et al. 2016. al. 2012. Type strain: strain Hauduroy L948 = ATCC Type strain: strain AB-18-032 = DSM 45365 = 19977 = DSM 44196 = CIP 104536 = JCM 31040 = NRRL B-24793 = NBRC CCUG 20993 = JCM 13569 = NCTC 13031. 109089 = TBRC 6556. Reference: Front Microbiol., 2018, 9: 67; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 181 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417]. Microbiol., 2018, 68: 3379–3393]. Mycobacteroides abscessus subsp. bolletii Micromonospora phaseoli (Wang et al. 2013) (Adékambi et al. 2006) Gupta et al. 2018, Nouioui et al. 2018, comb. nov. comb. nov. Basonym: Xiangella phaseoli Wang et al. Basonym: Mycobacterium abscessus subsp. 2013. bolletii (Adékambi et al. 2006) Leao et al. Type strain: strain NEAU-J5 = DSM 45730 = 2011 emend. Tortoli et al. 2016. NBRC 110907 = CGMCC 4.7038. Type strain: strain BD = CCUG 50184 = JCM Reference: Front Microbiol., 2018, 9: 2007; 15297 = CIP 108541. Validation List no. 184 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 67; Microbiol., 2018, 68: 3379–3393]. Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Micromonospora qiuiae (Xi et al. 2012) Nouioui et al. 2018, comb. nov. Mycobacteroides abscessus subsp. Basonym: Verrucosispora qiuiae Xi et al. massiliense (Adékambi et al. 2006) Gupta et 2012. al. 2018, comb. nov. Type strain: strain RtIII47 = DSM 45781 = Basonym: Mycobacterium abscessus subsp. JCM 19682 = BCC 51292 = CGMCC massiliense (Adékambi et al. 2006) Tortoli et 4.5826 = NBRC 106684. al. 2016. Reference: Front Microbiol., 2018, 9: 2007; Type strain: CCUG 48898 = DSM 45103 = CIP Validation List no. 184 [Int J Syst Evol 108297 = KCTC 19086. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycobacteroides abscessus (Moore and Microbiol., 2018, 68: 1411–1417] Frerichs 1953) Gupta et al. 2018, comb. nov. Basonym: Mycobacterium abscessus (Moore Mycobacteroides chelonae (Bergey et al. and Frerichs 1953) Kusunoki and Ezaki 1923) Gupta et al. 2018, comb. nov.

S28 Basonym: Mycobacterium chelonae corrig. Bergey et al. 1923 emend. Kim et al. 2017. Mycolicibacillus koreensis (Kim et al. 2012) Type strain: strain CM 6388 = ATCC 35752 = Gupta et al. 2018, comb. nov. DSM 43804 = CIP 104535 = CCUG 47445 Basonym: Mycobacterium koreense Kim et al. = JCM 6388. 2012. Reference: Front Microbiol., 2018, 9: 67; Type strain: strain 01–305 = DSM 45576 = Validation List no. 181 [Int J Syst Evol KCTC 19819. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycobacteroides franklinii (Nogueira et al. Microbiol., 2018, 68: 1411–1417] 2015) Gupta et al. 2018, comb. nov. Basonym: Mycobacterium franklinii Nogueira Mycolicibacillus parakoreensis (Kim et al. et al. 2015. 2013) Gupta et al. 2018, comb. nov. Type strain: DSM 45524 = ATCC BAA-2149. Basonym: Mycobacterium parakoreense Kim Reference: Front Microbiol., 2018, 9: 67; et al. 2013. Validation List no. 181 [Int J Syst Evol Type strain: strain 299 = DSM 45575 = KCTC Microbiol., 2018, 68: 1411–1417] 19818. Reference: Front Microbiol., 2018, 9: 67; Mycobacteroides immunogenum (Wilson et Validation List no. 181 [Int J Syst Evol al. 2001) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium immunogenum Wilson et al. 2001. Mycolicibacillus trivialis (Kubica et al. 1970) Type strain: strain BH29 = ATCC 700505 = Gupta et al. 2018, comb. nov. DSM 45595. Basonym: Mycobacterium triviale Kubica et al. Reference: Front Microbiol., 2018, 9: 67; 1970. Validation List no. 181 [Int J Syst Evol Type strain: ATCC 23292 = DSM 44153 = Microbiol., 2018, 68: 1411–1417]. CCUG 42431. Reference: Front Microbiol., 2018, 9: 67; Mycobacteroides salmoniphilum (Whipps et Validation List no. 181 [Int J Syst Evol al. 2007) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417]. Basonym: Mycobacterium salmoniphilum (ex Ross 1960) Whipps et al. 2007. Mycolicibacter algericus (Sahraoui et al. 2011) Type strain: strain SC = ATCC 13758 = DSM Gupta et al. 2018, comb. nov. 43276. Basonym: Mycobacterium algericum Sahraoui Reference: Front Microbiol., 2018, 9: 67; et al. 2011. Validation List no. 181 [Int J Syst Evol Type strain: strain TBE 500028/10 = strain Microbiol., 2018, 68: 1411–1417] Bejaia = CIP 110121 = DSM 45454. Reference: Front Microbiol., 2018, 9: 67; Mycobacteroides saopaulense (Nogueira et al. Validation List no. 181 [Int J Syst Evol 2015) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium saopaulense Nogueira et al. 2015. Mycolicibacter arupensis (Cloud et al. 2006) Type strain: strain EPM 10906 = CCUG 66554 Gupta et al. 2018, comb. nov. = LMG 28586 = INCQS 0733. Basonym: Mycobacterium arupense Cloud et Reference: Front Microbiol., 2018, 9: 67; al. 2006. Validation List no. 181 [Int J Syst Evol Type strain: strain AR30097 = ATCC BAA- Microbiol., 2018, 68: 1411–1417] 1242 = DSM 44942.

S29 Reference: Front Microbiol., 2018, 9: 67; Type strain: strain FI-07034 = CCUG 58460 = Validation List no. 181 [Int J Syst Evol DSM 45394. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycolicibacter engbaekii (Tortoli et al. 2013) Microbiol., 2018, 68: 1411–1417] Gupta et al. 2018, comb. nov. Basonym: Mycobacterium engbaekii Tortoli et Mycolicibacter minnesotensis (Hannigan et al. al. 2013. 2013) Gupta et al. 2018, comb. nov. Type strain: ATCC 27353 = DSM 45694. Basonym: Mycobacterium minnesotense Reference: Front Microbiol., 2018, 9: 67; Hannigan et al. 2013. Validation List no. 181 [Int J Syst Evol Type strain: strain DL49 = DSM 45633 = JCM Microbiol., 2018, 68: 1411–1417] 17932 = NCCB 100399. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacter heraklionensis (Tortoli et al. Validation List no. 181 [Int J Syst Evol 2013) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium heraklionense Tortoli et al. 2013. Mycolicibacter nonchromogenicus Type strain: strain GN-1 = CECT 7509 = LMG (Tsukamura 1965) Gupta et al. 2018, comb. 24735 = NCTC 13432. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium nonchromogenicum Validation List no. 181 [Int J Syst Evol Tsukamura 1965. Microbiol., 2018, 68: 1411–1417] Type strain: ATCC 19530 = DSM 44164 = JCM 6364 = CCUG 28009=CIP Mycolicibacter hiberniae (Kazda et al. 1993) 106811=NCTC 10424. Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium hiberniae Kazda et Validation List no. 181 [Int J Syst Evol al. 1993. Microbiol., 2018, 68: 1411–1417] Type strain: strain Hi 11 = ATCC 49874 = DSM 44241 = CIP 104537=JCM 13571. Mycolicibacter paraterrae (Lee et al. 2016) Reference: Front Microbiol., 2018, 9: 67; Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium paraterrae Lee et Microbiol., 2018, 68: 1411–1417]. al. 2016. Type strain: strain 05–2522 = DSM 45127 = Mycolicibacter kumamotonensis (Masaki et KCTC 19556. al. 2007) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium kumamotonense Validation List no. 181 [Int J Syst Evol Masaki et al. 2007. Microbiol., 2018, 68: 1411–1417]. Type strain: strain CST 7247 = CCUG 51961 = DSM 45093 = JCM 13453. Mycolicibacter senuensis (Mun et al. 2008) Reference: Front Microbiol., 2018, 9: 67; Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium senuense Mun et al. Microbiol., 2018, 68: 1411–1417] 2008. Type strain: strain 05–832 = DSM 44999 = Mycolicibacter longobardus (Tortoli et al. JCM 16017 = KCTC 1914. 2013) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium longobardum Validation List no. 181 [Int J Syst Evol Tortoli et al. 2013. Microbiol., 2018, 68: 1411–1417]

S30 Type strain: strain QIA-38 = JCM 30275 = Mycolicibacter terrae (Wayne 1966) Gupta et KCTC 29443. al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium terrae Wayne 1966. Validation List no. 181 [Int J Syst Evol Type strain: ATCC 15755 = DSM 43227 = Microbiol., 2018, 68: 1411–1417] CCUG 27847 = CIP 104321 = JCM 12143 = LMG 10394. Mycolicibacterium arabiense (Zhang et al. Reference: Front Microbiol., 2018, 9: 67; 2013) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium arabiense Zhang et Microbiol., 2018, 68: 1411–1417] al. 2013. Type strain: YIM 121001 = DSM 45768 = Mycolicibacterium agri (Tsukamura 1981) JCM 18538. Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium agri (ex Tsukamura Validation List no. 181 [Int J Syst Evol 1972) Tsukamura 1981. Microbiol., 2018, 68: 1411–1417] Type strain: strain 90012 = ATCC 27406 = DSM 44515 = CCUG 37673 A = CIP Mycolicibacterium arcueilense (Konjek et al. 105391. 2016) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium arcueilense Konjek Validation List no. 181 [Int J Syst Evol et al. 2016. Microbiol., 2018, 68: 1411–1417] Type strain: strain 269 = ParisRGMnew_3 = CIP 110654 = DSM 46715. Mycolicibacterium aichiense (Tsukamura et Reference: Front Microbiol., 2018, 9: 67; al. 1981) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium aichiense (ex Microbiol., 2018, 68: 1411–1417] Tsukamura 1973) Tsukamura 1981. Type strain: strain 49 005 = ATCC 27280 = Mycolicibacterium aromaticivorans DSM 44147 = CIP 106808 = JCM 6376 = (Hennessee et al. 2009) Gupta et al. 2018, LMG 19259 = NCTC 10820. comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium aromaticivorans Validation List no. 181 [Int J Syst Evol Hennessee et al. 2009. Microbiol., 2018, 68: 1411–1417] Type strain: strain JS19b1 = ATCC BAA-1378 = JCM 16368 = CIP 109274. Mycolicibacterium alvei (Ausina et al. 1992) Reference: Front Microbiol., 2018, 9: 67; Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium alvei Ausina et al. Microbiol., 2018, 68: 1411–1417] 1992. Type strain: strain CR-21 = ATCC 51304 = Mycolicibacterium aubagnense (Adékambi et JCM 12272 = CIP 103464 = DSM 44176. al. 2006) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium aubagnense Validation List no. 181 [Int J Syst Evol Adékambi et al. 2006. Microbiol., 2018, 68: 1411–1417]. Type strain: strain U8 = CCUG 50186 = JCM 15296 = CIP 108543. Mycolicibacterium anyangense (Kim et al. Reference: Front Microbiol., 2018, 9: 67; 2015) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium anyangense Kim et Microbiol., 2018, 68: 1411–1417]. al. 2015.

S31 Mycolicibacterium aurum (Tsukamura 1966) Schinsky et al. 2004. Gupta et al. 2018, comb. nov. Type strain: strain W6743 = ATCC 49938 = Basonym: Mycobacterium aurum Tsukamura et DSM 44680 = CCUG 47584 = JCM 15654. al. 1966. Reference: Front Microbiol., 2018, 9: 67; Type strain: ATCC 23366 = DSM 43999 = Validation List no. 181 [Int J Syst Evol CCUG 37666 = CIP 104465 = HAMBI 2275 Microbiol., 2018, 68: 1411–1417]. = JCM 6366 = LMG 19255 = NCTC 10437 = NRRL B-4037. Mycolicibacterium brumae (Luquin et al. Reference: Front Microbiol., 2018, 9: 67; 1993) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium brumae Luquin et Microbiol., 2018, 68: 1411–1417] al. 1993. Type strain: strain CR-270 = ATCC 51384 = Mycolicibacterium austroafricanum DSM 44177 = CCUG 37586 = CIP 103465 (Tsukamura 1983) Gupta et al. 2018, comb. = JCM 12273. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium austroafricanum Validation List no. 181 [Int J Syst Evol Tsukamura 1983. Microbiol., 2018, 68: 1411–1417] Type strain: strain E9789-SA12441 = ATCC 33464 = DSM 44191 = CCUG 37667 = CIP Mycolicibacterium canariasense (Jiménez et 105395 = HAMBI 2271 = JCM 6369. al. 2004) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium canariasense Validation List no. 181 [Int J Syst Evol Jiménez et al. 2004. Microbiol., 2018, 68: 1411–1417] Type strain: strain 502329 = CCUG 47953 = JCM 15298 = CIP 107998. Mycolicibacterium bacteremicum (Brown- Reference: Front Microbiol., 2018, 9: 67; Elliott et al. 2012) Gupta et al. 2018, comb. Validation List no. 181 [Int J Syst Evol nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium bacteremicum Brown-Elliott et al. 2012. Mycolicibacterium celeriflavum (Shahraki et Type strain: ATCC 25791 = DSM 45578. al. 2015) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium celeriflavum Validation List no. 181 [Int J Syst Evol Shahraki et al. 2015. Microbiol., 2018, 68: 1411–1417] Type strain: strain AFPC-000207 = DSM 46765 = JCM 18439. Mycolicibacterium boenickei (Schinsky et al. Reference: Front Microbiol., 2018, 9: 67; 2004) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium boenickei Schinsky Microbiol., 2018, 68: 1411–1417] et al. 2004. Type strain: strain W5998 = ATCC 49935 = Mycolicibacterium chitae (Tsukamura 1967) DSM 44677 = JCM 15653. Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium chitae Tsukamura Validation List no. 181 [Int J Syst Evol 1967. Microbiol., 2018, 68: 1411–1417] Type strain: ATCC 19627 = DSM 44633 = CCUG 39504 = CIP 105383 = JCM 12403 = Mycolicibacterium brisbanense (Schinsky et NCTC 10485. al. 2004) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium brisbanense Validation List no. 181 [Int J Syst Evol

S32 Microbiol., 2018, 68: 1411–1417] Microbiol., 2018, 68: 1411–1417]

Mycolicibacterium chlorophenolicum Mycolicibacterium cosmeticum (Cooksey et [(Apajalathi et al. 1986) Häggblom et al. al. 2004) Gupta et al. 2018, comb. nov. 1994] Gupta et al. 2018, comb. nov. Basonym: Mycobacterium cosmeticum Basonym: Mycobacterium chlorophenolicum Cooksey et al. 2004. (Apajalathi et al. 1986) Häggblom et al. Type strain: strain LTA-388 = ATCC BAA-878 1994. = JCM 14739 = CIP 108170. Type strain: strain PCP-I = ATCC 49826 = Reference: Front Microbiol., 2018, 9: 67; DSM 43826 = CIP 104189 = HAMBI 2278 Validation List no. 181 [Int J Syst Evol = IEGM 559 = NBRC 15527 = JCM 7439 = Microbiol., 2018, 68: 1411–1417] NRRL B-16528. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium crocinum (Hennessee et al. Validation List no. 181 [Int J Syst Evol 2009) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417]. Basonym: Mycobacterium crocinum Hennessee et al. 2009. Mycolicibacterium chubuense (Tsukamura Type strain: strain czh-42 = ATCC BAA-1373 1981) Gupta et al. 2018, comb. nov. = JCM 16369 = CIP 109269. Basonym: Mycobacterium chubuense (ex Reference: Front Microbiol., 2018, 9: 67; Tsukamura 1973) Tsukamura 1981. Validation List no. 181 [Int J Syst Evol Type strain: strain 48 013 (previously, strain Microbiol., 2018, 68: 1411–1417] 5517) = ATCC 27278 = DSM 44219 = CCUG 37670 = CIP 106810 = JCM 6374 = Mycolicibacterium diernhoferi (Tsukamura et NCTC 10819. al. 1983) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium diernhoferi (ex Validation List no. 181 [Int J Syst Evol Bönicke and Juhasz 1965) Tsukamura et al. Microbiol., 2018, 68: 1411–1417] 1983. Type strain: strain 41 001 = ATCC 19340 = Mycolicibacterium conceptionense DSM 43524 = CIP 105384 = HAMBI 2269 (Adékambiet al. 2006) Gupta et al. 2018, = NBRC 14756 = JCM 6371. comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium conceptionense Validation List no. 181 [Int J Syst Evol Adékambiet et al. 2006. Microbiol., 2018, 68: 1411–1417]. Type strain: strain D16 = CCUG 50187 = JCM 15299 = CIP 108544. Mycolicibacterium doricum (Tortoli et al. Reference: Front Microbiol., 2018, 9: 67; 2001) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium doricum Tortoli et Microbiol., 2018, 68: 1411–1417] al. 2001. Type strain: strain FI-13295 = DSM 44339 = Mycolicibacterium confluentis (Kirschner et JCM 12405 = CCUG 46352 = CIP 106867. al. 1992) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium confluentis Validation List no. 181 [Int J Syst Evol Kirschner et al. 1992. Microbiol., 2018, 68: 1411–1417] Type strain: strain 1389/90 = ATCC 49920 = DSM 44017 = CIP 105510 = JCM 13671. Mycolicibacterium duvalii (Stanford and Reference: Front Microbiol., 2018, 9: 67; Gunthorpe 1971) Gupta et al. 2018, comb. Validation List no. 181 [Int J Syst Evol nov.

S33 Basonym: Mycobacterium duvalii Stanford and Reference: Front Microbiol., 2018, 9: 67; Gunthorpe 1971. Validation List no. 181 [Int J Syst Evol Type strain: ATCC 43910 = DSM 44244 = Microbiol., 2018, 68: 1411–1417] CCUG 41352 = CIP 104539 = JCM 6396 = NCTC 358. Mycolicibacterium fluoranthenivorans Reference: Front Microbiol., 2018, 9: 67; (Hormisch et al. 2006) Gupta et al. 2018, Validation List no. 181 [Int J Syst Evol comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium fluoranthenivorans Hormisch et al. 2006. Mycolicibacterium elephantis (Shojaei et al. Type strain: strain FA4 = DSM 44556 = JCM 2000) Gupta et al. 2018, comb. nov. 14741 = CIP 108203. Basonym: Mycobacterium elephantis Shojaei Reference: Front Microbiol., 2018, 9: 67; et al. 2000. Validation List no. 181 [Int J Syst Evol Type strain: strain 484 = DSM 44368 = JCM Microbiol., 2018, 68: 1411–1417] 12406 = CIP 106831. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium fortuitum (da Costa Cruz Validation List no. 181 [Int J Syst Evol 1938) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium fortuitum da Costa Cruz 1938. Mycolicibacterium fallax (Lévy-Frébault et al. Type strain: ATCC 6841 = DSM 46621 = 1983) Gupta et al. 2018, comb. nov. CCUG 20994 = CIP 104534 = NBRC 13159 Basonym: Mycobacterium fallax Lévy-Frébault = JCM 6387 = NCTC 10394. et al. 1983. Reference: Front Microbiol., 2018, 9: 67; Type strain: ATCC 35219 = DSM 44179 = Validation List no. 181 [Int J Syst Evol CCUG 37584 = CIP 81.39 = JCM 6405. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycolicibacterium fortuitum subsp. Microbiol., 2018, 68: 1411–1417] acetamidolyticum (Tsukuamura et al. 1986) Gupta et al. 2018, comb. nov. Mycolicibacterium farcinogenes (Chamoiseau Basonym: Mycobacterium fortuitum subsp. 1973) Gupta et al. 2018, comb. nov. acetamidolyticum Tsukuamura et al. 1986. Basonym: Mycobacterium farcinogenes Type strain: strain NCH E11620 = DSM 44220 Chamoiseau 1973. = JCM 6368 = ATCC 35931 = CIP 105423. Type strain: strain IEMVT 75 = ATCC 35753 = Reference: Front Microbiol., 2018, 9: 67; DSM 43637 = CCUG 21047 = JCM 15463 = Validation List no. 181 [Int J Syst Evol NCTC 10955. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycolicibacterium fortuitum subsp. fortuitum Microbiol., 2018, 68: 1411–1417]. [(da Costa Cruz 1938) Tsukuamura et al. 1986] Gupta et al. 2018, comb. nov. Mycolicibacterium flavescens (Bojalil et al. Basonym: Mycobacterium fortuitum subsp. 1962) Gupta et al. 2018, comb. nov. fortuitum (da Costa Cruz 1938) Tsukuamura Basonym: Mycobacterium flavescens Bojalil et et al. 1986. al. 1962. Type strain: ATCC 6841 = DSM 46621 = Type strain: ATCC 14474 = DSM 43991 = CCUG 20994 = CIP 104534 = NBRC 13159 CCUG 29041 = CIP 104533 = JCM 12274 = = JCM 6387 = NCTC 10394. NCTC 10271 = NRRL B-4038. Reference: Front Microbiol., 2018, 9: 67;

S34 Validation List no. 181 [Int J Syst Evol 2016) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417]. Basonym: Mycobacterium helvum Tran and Dahl 2016. Mycolicibacterium frederiksbergense Type strain: strain DL739 = JCM 30396 = (Willumsen et al. 2001) Gupta et al. 2018, NCCB 100520. comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium frederiksbergense Validation List no. 181 [Int J Syst Evol Willumsen et al. 2001. Microbiol., 2018, 68: 1411–1417]. Type strain: strain FAn9 = DSM 44346 = NRRL B-24126 = CIP 107205. Mycolicibacterium hippocampi (Balcázar et Reference: Front Microbiol., 2018, 9: 67; al. 2014) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium hippocampi Microbiol., 2018, 68: 1411–1417] Balcázar et al. 2014. Type strain: strain BFLP-6 = DSM 45391 = Mycolicibacterium gadium (Casal and Calero LMG 25372. 1974) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium gadium Casal and Validation List no. 181 [Int J Syst Evol Calero 1974 Microbiol., 2018, 68: 1411–1417] Type strain: ATCC 27726 = DSM 44077 = CCUG 37515 = CIP 105388 = HAMBI 2274 Mycolicibacterium hodleri (Kleespies et al. = JCM 12688 = NCTC 10942. 1996) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium hodleri Kleespies et Validation List no. 181 [Int J Syst Evol al. 1996. Microbiol., 2018, 68: 1411–1417] Type strain: strain EM12 = DSM 44183 = JCM 12141 = CIP 104909 = LMG 19253. Mycolicibacterium gilvum (Stanford and Reference: Front Microbiol., 2018, 9: 67; Gunthorpe 1971) Gupta et al. 2018, comb. Validation List no. 181 [Int J Syst Evol nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium gilvum Stanford and Gunthorpe 1971. Mycolicibacterium holsaticum (Richter et al. Type strain: ATCC 43909 = JCM 15464 = CIP 2002) Gupta et al. 2018, comb. nov. 106743 = NCTC 10742. Basonym: Mycobacterium holsaticum Reference: Front Microbiol., 2018, 9: 67; Richteret al. 2002. Validation List no. 181 [Int J Syst Evol Type strain: strain 1406 = DMS 44478 = JCM Microbiol., 2018, 68: 1411–1417] 12374 = CCUG 46266. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium hassiacum (Schröder et al. Validation List no. 181 [Int J Syst Evol 1997) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium hassiacum Schröder et al. 1997. Mycolicibacterium houstonense (Schinsky et Type strain: strain 3849 = DSM 44199 = JCM al. 2004) Gupta et al. 2018, comb. nov. 12690 = CCUG 37519 = JCM 12690. Basonym: Mycobacterium houstonense Reference: Front Microbiol., 2018, 9: 67; Schinsky et al. 2004. Validation List no. 181 [Int J Syst Evol Type strain: strain W5198 = ATCC 49403 = Microbiol., 2018, 68: 1411–1417] DSM 44676 = JCM 15656. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium helvum (Tran and Dahl Validation List no. 181 [Int J Syst Evol

S35 Microbiol., 2018, 68: 1411–1417] Type strain: strain MG13 = CCUG 54744 = JCM 16229 = CECT 7273. Mycolicibacterium insubricum (Tortoli et al. Reference: Front Microbiol., 2018, 9: 67; 2009) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium insubricum Tortoli Microbiol., 2018, 68: 1411–1417] et al. 2009. Type strain: strain FI-06250 = DSM 45132 = Mycolicibacterium lutetiense (Konjek et al. JCM 16366 = CIP 109609. 2016) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium lutetiense Konjek et Validation List no. 181 [Int J Syst Evol al. 2016. Microbiol., 2018, 68: 1411–1417]. Type strain: strain 071 = strain ParisRGMnew_1 = CIP 110656 = DSM Mycolicibacterium iranicum (Shojaei et al. 46713. 2013) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium iranicum Shojaei et Validation List no. 181 [Int J Syst Evol al. 2013. Microbiol., 2018, 68: 1411–1417]. Type strain: strain M05 = DSM 45541 = JCM 17461 = CCUG 62053. Mycolicibacterium madagascariense (Kazda Reference: Front Microbiol., 2018, 9: 67; et al. 1992) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium madagascariense Microbiol., 2018, 68: 1411–1417] Kazda et al. 1992. Type strain: strain P2 = ATCC 49865 = JCM Mycolicibacterium komossense (Kazda and 13574 = CIP 104538. Müller 1979) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium komossense Kazda Validation List no. 181 [Int J Syst Evol and Müller 1979. Microbiol., 2018, 68: 1411–1417] Type strain: strain Ko 2 = ATCC 33018 = DSM 44078 = CIP 105293 = HAMBI 2279 = Mycolicibacterium mageritense (Domenech et HAMBI 2280 = JCM 12408. al. 1997) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium mageritense Validation List no. 181 [Int J Syst Evol Domenech et al. 1997. Microbiol., 2018, 68: 1411–1417] Type strain: strain 938 = ATCC 700351 = DSM 44476 = CCUG 37984 = CIP 104973 = JCM Mycolicibacterium litorale (Zhang et al. 2012) 12375. Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium litorale Zhang et al. Validation List no. 181 [Int J Syst Evol 2012. Microbiol., 2018, 68: 1411–1417] Type strain: strain F4 = DSM 45785 = JCM 17423 = CGMCC 4.5724. Mycolicibacterium malmesburyense (Gcebe et Reference: Front Microbiol., 2018, 9: 67; al. 2017) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium malmesburyense Microbiol., 2018, 68: 1411–1417] Gcebe et al. 2017. Type strain: strain WCM 7299 = ATCC BAA- Mycolicibacterium llatzerense (Gomila et al. 2759 = CIP 110822. 2008) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium llatzerense Gomila Validation List no. 181 [Int J Syst Evol et al. 2008. Microbiol., 2018, 68: 1411–1417]

S36 Type strain: ATCC 25795 = DSM 44074 = Mycolicibacterium monacense (Reischl et al. CCUG 37665 = CIP 105387 = HAMBI 2273 2006) Gupta et al. 2018, comb. nov. = JCM 6365 = NCTC 10818. Basonym: Mycobacterium monacense Reischl Reference: Front Microbiol., 2018, 9: 67; et al. 2006. Validation List no. 181 [Int J Syst Evol Type strain: strain B9-21-178 = DSM 44395 = Microbiol., 2018, 68: 1411–1417] JCM 15658 = CIP 109237. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium neworleansense (Schinsky Validation List no. 181 [Int J Syst Evol et al. 2004) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium neworleansense Schinsky et al. 2004. Mycolicibacterium moriokaense (Tsukamura Type strain: strain W6705 = ATCC 49404 = et al. 1986) Gupta et al. 2018, comb. nov. DSM 44679 = JCM 15659. Basonym: Mycobacterium moriokaense Reference: Front Microbiol., 2018, 9: 67; Tsukamura et al. 1986. Validation List no. 181 [Int J Syst Evol Type strain: strain NCH E11715 = ATCC Microbiol., 2018, 68: 1411–1417] 43059 = DSM 44221 = CCUG 37671 = CIP 105393 = JCM 6375 = VKM Ac-1183. Mycolicibacterium novocastrense (Shojaei et Reference: Front Microbiol., 2018, 9: 67; al. 1997) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium novocastrense Microbiol., 2018, 68: 1411–1417]. Shojaei et al. 1997. Type strain: strain 73 = DSM 44203 = JCM Mycolicibacterium mucogenicum (Springer et 18114 = CIP 105546. al. 1995) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium mucogenicum Validation List no. 181 [Int J Syst Evol Springer et al. 1995. Microbiol., 2018, 68: 1411–1417]. Type strain: strain MO76 = ATCC 49650 = CCUG 47451. Mycolicibacterium obuense (Tsukamura et al. Reference: Front Microbiol., 2018, 9: 67; 1981) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium obuense (ex Microbiol., 2018, 68: 1411–1417] Tsukamura and Mizuno 1971) Tsukamura et al. 1981. Mycolicibacterium murale (Vuorio et al. 1999) Type strain: strain 47 001 (previously, strain Gupta et al. 2018, comb. nov. 4388) = ATCC 27023 = DSM 44075 = Basonym: Mycobacterium murale Vuorio et al. CCUG 37669 = CIP 106803 = HAMBI 2272 1999. = JCM 6372 = NCTC 10778. Type strain: strain MA112/96 = CCUG 39728 Reference: Front Microbiol., 2018, 9: 67; = DSM 44340 = CIP 105980 = HAMBI Validation List no. 181 [Int J Syst Evol 2320 = JCM 13392. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycolicibacterium oryzae (Ramaprasad et al. Microbiol., 2018, 68: 1411–1417] 2016) Gupta et al. 2018, comb. nov. Basonym: Mycobacterium oryzae Ramaprasad Mycolicibacterium neoaurum (Tsukamura et al. 2016. 1972) Gupta et al. 2018, comb. nov. Type strain: strain JC290 = KCTC 39560 = Basonym: Mycobacterium neoaurum LMG 28809. Tsukamura 1972. Reference: Front Microbiol., 2018, 9: 67;

S37 Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Mycolicibacterium phocaicum (Adékambi et al. 2006) Gupta et al. 2018, comb. nov. Mycolicibacterium pallens (Hennessee et al. Basonym: Mycobacterium phocaicum 2009) Gupta et al. 2018, comb. nov. Adékambi et al. 2006. Basonym: Mycobacterium pallens Hennessee Type strain: strain N4 = CCUG 50185 = JCM et al. 2009. 15301 = CIP 108542. Type strain: strain czh-8 = ATCC BAA-1372 = Reference: Front Microbiol., 2018, 9: 67; JCM 16370 = CIP 109268. Validation List no. 181 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 67; Microbiol., 2018, 68: 1411–1417] Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Mycolicibacterium porcinum (Tsukamura et al. 1983) Gupta et al. 2018, comb. nov. Mycolicibacterium parafortuitum (Tsukamura Basonym: Mycobacterium porcinum 1965) Gupta et al. 2018, comb. nov. Tsukamura et al. 1983. Basonym: Mycobacterium parafortuitum Type strain: strain E10241-1 = ATCC 33776 = Tsukamura 1965. DSM 44242 = CCUG 37674 = CIP 105392 Type strain: ATCC 19686 = DSM 43528 = = JCM 6378. CCUG 20999 = CIP 106802 = JCM 6367 = Reference: Front Microbiol., 2018, 9: 67; NCTC 10411 = NRRL B-4035. Validation List no. 181 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 67; Microbiol., 2018, 68: 1411–1417] Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Mycolicibacterium poriferae (Padgitt and Moshier 1987) Gupta et al. 2018, comb. nov. Mycolicibacterium peregrinum (Kusunoki and Basonym: Mycobacterium poriferae Padgitt Ezaki 1992) Gupta et al. 2018, comb. nov. and Moshier 1987. Basonym: Mycobacterium peregrinum (ex Type strain: strain no. 47 = ATCC 35087 = Bojalil et al. 1962) Kusunoki and Ezaki JCM 12603 = CIP 105394. 1992. Reference: Front Microbiol., 2018, 9: 67; Type strain: ATCC 14467 = JCM 12142 = Validation List no. 181 [Int J Syst Evol CCUG 27976 = CIP 105382 = NCTC 10264. Microbiol., 2018, 68: 1411–1417] Reference: Front Microbiol., 2018, 9: 67; Validation List no. 181 [Int J Syst Evol Mycolicibacterium psychrotolerans (Trujillo et Microbiol., 2018, 68: 1411–1417]. al. 2004) Gupta et al. 2018, comb. nov. Basonym: Mycobacterium psychrotolerans Mycolicibacterium phlei (Lehmann and Trujillo et al. 2004. Neumann 1899) Gupta et al. 2018, comb. Type strain: strain WA101 = DSM 44697 = nov. JCM 13323 = LMG 21953. Basonym: Mycobacterium phlei Lehmann and Reference: Front Microbiol., 2018, 9: 67; Neumann 1899. Validation List no. 181 [Int J Syst Evol Type strain: ATCC 11758 = DSM 43239 = Microbiol., 2018, 68: 1411–1417]. JCM 5865 = CCUG 21000 = CIP 105389 = JCM 6385 = NCTC 8151 = NRRL B-14615 Mycolicibacterium pulveris (Tsukamura et al. = VKM Ac-1291. 1983) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium pulveris Tsukamura Validation List no. 181 [Int J Syst Evol et al. 1983. Microbiol., 2018, 68: 1411–1417] Type strain: strain NCH 33505 = ATCC 35154

S38 = DSM 44222 = CCUG 37668 = CIP 106804 = JCM 6370. Mycolicibacterium sarraceniae (Tran and Reference: Front Microbiol., 2018, 9: 67; Dahl 2016) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium sarraceniae Tran Microbiol., 2018, 68: 1411–1417] and Dahl 2016. Type strain: strain DL734 = JCM 30395 = Mycolicibacterium pyrenivorans (Derz et al. NCCB 100519. 2004) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium pyrenivorans Derz Validation List no. 181 [Int J Syst Evol et al. 2004. Microbiol., 2018, 68: 1411–1417] Type strain: strain 17A3 = DSM 44605 = JCM 15927 = NRRL B-24349. Mycolicibacterium sediminis (Zhang et al. Reference: Front Microbiol., 2018, 9: 67; 2013) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium sediminis Zhang et Microbiol., 2018, 68: 1411–1417] al. 2013. Type strain: YIM M13028 = DSM 45643 = Mycolicibacterium rhodesiae (Tsukamura et KCTC 19999. al. 1981) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium rhodesiae (ex Validation List no. 181 [Int J Syst Evol Tsukamura et al. 1971) Tsukamura et al. Microbiol., 2018, 68: 1411–1417] 1981. Type strain: strain 02 002 (previously, strain Mycolicibacterium senegalense (Chamoiseau 5295) = ATCC 27024 = DSM 44223 = CIP 1973) Gupta et al. 2018, comb. nov. 106806 = JCM 6363 = NCTC 10779. Basonym: Mycobacterium senegalense Reference: Front Microbiol., 2018, 9: 67; (Chamoiseau 1973) Chamoiseau 1979. Validation List no. 181 [Int J Syst Evol Type strain: strain IEMVT 378 = ATCC 35796 Microbiol., 2018, 68: 1411–1417] = DSM 43656 = CCUG 21001 = CIP 104941 = JCM 15467 = NCTC 10956. Mycolicibacterium rufum (Hennessee et al. Reference: Front Microbiol., 2018, 9: 67; 2009) Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium rufum Hennessee et Microbiol., 2018, 68: 1411–1417] al. 2009. Type strain: strain JS14 = ATCC BAA-1377 = Mycolicibacterium septicum (Schinsky et al. JCM 16372 = CIP 109273. 2000) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium septicum Schinsky Validation List no. 181 [Int J Syst Evol et al. 2000. Microbiol., 2018, 68: 1411–1417] Type strain: strain W4964 = ATCC 700731 = DSM 44393 = CCUG 43574 = CIP 106642 Mycolicibacterium rutilum (Hennessee et al. = JCM 14743. 2009) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Mycobacterium rutilum Hennessee Validation List no. 181 [Int J Syst Evol et al. 2009. Microbiol., 2018, 68: 1411–1417] Type strain: strain czh-117 = ATCC BAA-1375 = JCM 16371 = CIP 109271. Mycolicibacterium setense (Lamy et al. 2008) Reference: Front Microbiol., 2018, 9: 67; Gupta et al. 2018, comb. nov. Validation List no. 181 [Int J Syst Evol Basonym: Mycobacterium setense Lamy et al. Microbiol., 2018, 68: 1411–1417]. 2008.

S39 Type strain: strain ABO-M06 = DSM 45070 = Reference: Front Microbiol., 2018, 9: 67; JCM 15660 = CIP 109395. Validation List no. 181 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 67; Microbiol., 2018, 68: 1411–1417] Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Mycolicibacterium tusciae (Tortoli et al. 1999) Gupta et al. 2018, comb. nov. Mycolicibacterium smegmatis (Trevisan 1889) Basonym: Mycobacterium tusciae Tortoli et al. Gupta et al. 2018, comb. nov. 1999. Basonym: Mycobacterium smegmatis (Trevisan Type strain: strain FI-25796 = DSM 44338 = 1889) Lehmann and Neumann 1899. JCM 12692 = CCUG 50996 = CIP 106367. Type strain: ATCC 19420 = DSM 43756 = Reference: Front Microbiol., 2018, 9: 67; CCUG 21002 = CCUG 21815 = CIP 104444 Validation List no. 181 [Int J Syst Evol = JCM 5866 = NCTC 8159 = NRRL B- Microbiol., 2018, 68: 1411–1417] 14616 = VKM Ac-1239. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium vaccae (Bönicke and Validation List no. 181 [Int J Syst Evol Juhasz 1964) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium vaccae Bönicke and Juhasz 1964. Mycolicibacterium sphagni (Kazda 1980) Type strain: ATCC 15483 = DSM 43292 = Gupta et al. 2018, comb. nov. CCUG 21003 = CIP 105934 = HAMBI 2276 Basonym: Mycobacterium sphagni Kazda = JCM 6389 = NBRC 14118 = NCTC 1980. 10916. Type strain: strain Sph 38 = ATCC 33027 = Reference: Front Microbiol., 2018, 9: 67; DSM 44076. Validation List no. 181 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 67; Microbiol., 2018, 68: 1411–1417] Validation List no. 181 [Int J Syst Evol Microbiol., 2018, 68: 1411–1417] Mycolicibacterium vanbaalenii (Khan et al. 2000) Gupta et al. 2018, comb. nov. Mycolicibacterium thermoresistibile Basonym: Mycobacterium vanbaalenii Khan et (Tsukamura 1966) Gupta et al. 2018, comb. al. 2000. nov. Type strain: strain PYR-1 = DSM 7251 = JCM Basonym: Mycobacterium thermoresistibile 13017 = NRRL B-24157. Tsukamura 1966. Reference: Front Microbiol., 2018, 9: 67; Type strain: ATCC 19527 = DSM 44167 = Validation List no. 181 [Int J Syst Evol CCUG 28008 = CCUG 41353 = CIP 105390 Microbiol., 2018, 68: 1411–1417] = JCM 6362 = NCTC 10409. Reference: Front Microbiol., 2018, 9: 67; Mycolicibacterium vulneris (van Ingen et al. Validation List no. 181 [Int J Syst Evol 2009) Gupta et al. 2018, comb. nov. Microbiol., 2018, 68: 1411–1417] Basonym: Mycobacterium vulneris van Ingen et al. 2009. Mycolicibacterium tokaiense (Tsukamura et Type strain: strain NLA000700772 = DSM al. 1981) Gupta et al. 2018, comb. nov. 45247 = JCM 18115 = CIP 109859. Basonym: Mycobacterium tokaiense (ex Reference: Front Microbiol., 2018, 9: 67; Tsukamura 1973) Tsukamura 1981. Validation List no. 181 [Int J Syst Evol Type strain: strain 47 503 (previously, strain Microbiol., 2018, 68: 1411–1417] 5553) = ATCC 27282 = DSM 44635 = CIP 106807 = JCM 6373 = NCTC 10821. Mycolicibacterium wolinskyi (Brown et al.

S40 1999) Gupta et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Mycobacterium wolinskyi Brown et Validation List no. 184 [Int J Syst Evol al. 1999. Microbiol., 2018, 68: 3379–3393]. Type strain: strain MO739 = ATCC 700010 = JCM 13393 = CCUG 47168 = CIP 106348 = Pedococcus cremeus (Zhang et al. 2011) DSM 44493. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 67; Basonym: Phycicoccus cremeus Zhang et al. Validation List no. 181 [Int J Syst Evol 2001. Microbiol., 2018, 68: 1411–1417] Type strain: strain V2M29 = DSM 28108 = JCM 17739 = CGMCC 1.6963 = NBRC Pauljensenia hongkongensis (Woo et al. 104261. 2004) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces hongkongensis Woo et Validation List no. 184 [Int J Syst Evol al. 2004. Microbiol., 2018, 68: 3379–3393]. Type strain: strain HKU8 = CCUG 48484 = DSM 15629 = CIP 107949 = LMG 21939. Pedococcus dokdonensis (Yoon et al. 2008) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Phycicoccus dokdonensis Yoon et Microbiol., 2018, 68: 3379–3393]. al. 2008. Type strain: strain DS-8 = DSM 22329 = JCM Pedococcus aerophilus (Weon et al. 2008) 19120 = KCTC 19248 = CCUG 54521. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Phycicoccus aerophilus Weon et al. Validation List no. 184 [Int J Syst Evol 2008. Microbiol., 2018, 68: 3379–3393]. Type strain: strain 5516T-20 = DSM 18548 = JCM 16378 = KACC 20658 = NBRC Pedococcus ginsenosidimutans (Wang et al. 106307. 2011) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Phycicoccus ginsenosidimutans Validation List no. 184 [Int J Syst Evol Wang et al. 2011. Microbiol., 2018, 68: 3379–3393]. Type strain: strain BXN5-13 = DSM 21006 = JCM 18961 = KCTC 19419. Pedococcus badiiscoriae (Lee 2013) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Phycicoccus badiiscoriae Lee 2013. Microbiol., 2018, 68: 3379–3393]. Type strain: strain Sco-B23 = DSM 23987 = NBRC 107918 = KCTC 19807 = KACC Pedococcus soli (Singh et al. 2015) Nouioui et 15111. al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Phycicoccus soli Singh et al. 2015. Validation List no. 184 [Int J Syst Evol Type strain: strain THG-a14 = DSM 103360 = Microbiol., 2018, 68: 3379–3393]. JCM 19837 = KACC 17892. Reference: Front Microbiol., 2018, 9: 2007; Pedococcus bigeumensis (Dastager et al. Validation List no. 184 [Int J Syst Evol 2008) Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Phycicoccus bigeumensis Dastager et al. 2003. Phycicoccus duodecadis (Lochhead 1958) Type strain: strain MSL-30 = DSM 19264 = Nouioui et al. 2018, comb. nov. JCM 16023 = KCTC 19266. Basonym: Arthrobacter duodecadis Lochhead

S41 1958. al. 2018, comb. nov. Type strain: ATCC 13347 = NBRC 12959 = Basonym: Micrococcus kristinae Kloos et al. NCIMB 9222. 1974. Reference: Front Microbiol., 2018, 9: 2007; Type strain: DSM 20032 = JCM 7237 = ATCC Validation List no. 184 [Int J Syst Evol 27570 = CCM 2690 = CCUG 33026 = CIP Microbiol., 2018, 68: 3379–3393]. 81.69 = IEGM 390 = LMG 14215 = NBRC 15354 = NCTC 11038 = NRRL B-14835 = Phycicoccus elongatus (Hanada et al. 2002) VKM B-1811. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Tetrasphaera elongata Hanada et al. Validation List no. 184 [Int J Syst Evol 2002. Microbiol., 2018, 68: 3379–3393]. Type strain: strain Lp2 = DSM 14184 = JCM 11141 = CCUG 46560 = CIP 107623 = Saccharomonospora iraqiensis (Ruan et al. NBRC 103079. 1994) Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinopolyspora iraqiensis Ruan et Validation List no. 184 [Int J Syst Evol al. 1994. Microbiol., 2018, 68: 3379–3393]. Type strain: strain IQ-H1 = DSM 44640 = JCM 9891 = BCRC 16263 = CGMCC 4.1193 = Rathayibacter agropyri (non O’Gara 1916) NBRC 103187. Schroeder et al. 2018, comb. nov., nom. rev. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Aplanobacter agropyri O’Gara Validation List no. 184 [Int J Syst Evol 1916. Microbiol., 2018, 68: 3379–3393]. Type strain: strain CA-4 = DSM 104101 = ATCC TSD-78. Schaalia canis (Hoyles et al. 2000) Nouioui et Reference: Int J Syst Evol Microbiol., 2018, al. 2018, comb. nov. 68: 1519–1525. Basonym: Actinomyces canis Hoyles et al. 2000. Rothia halotolerans (Tang et al. 2009) Type strain: strain M2289/98/2 = CCUG 41706 Nouioui et al. 2018, comb. nov. = DSM 15536. Basonym: Kocuria halotolerans Tang et al. Reference: Front Microbiol., 2018, 9: 2007; 2009. Validation List no. 184 [Int J Syst Evol Type strain: DSM 18442 = JCM 31975 = Microbiol., 2018, 68: 3379–3393]. CCTCC AB 206069 = KCTC 19172. Reference: Front Microbiol., 2018, 9: 2007; Schaalia cardiffensis (Hall et al. 2003) Validation List no. 184 [Int J Syst Evol Nouioui et al. 2018, comb. nov. Microbiol., 2018, 68: 3379–3393]. Basonym: Actinomyces cardiffensis Hall et al. 2003. Rothia koreensis (Park et al. 2010) Nouioui et Type strain: strain R10394 = CCUG 44997 = al. 2018, comb. nov. CIP 107323. Basonym: Kocuria koreensis Park et al. 2010. Reference: Front Microbiol., 2018, 9: 2007; Type strain: strain P31 = DSM 23367 = JCM Validation List no. 184 [Int J Syst Evol 15915 = KCTC 19595. Microbiol., 2018, 68: 3379–3393]. Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Schaalia funkei (Lawson et al. 2001) Nouioui Microbiol., 2018, 68: 3379–3393]. et al. 2018, comb. nov. Basonym: Actinomyces funkei Lawson et al. Rothia kristinae (Kloos et al. 1974) Nouioui et 2001.

S42 Type strain: CCUG 42773 = CIP 106731. al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces odontolyticus Batty Validation List no. 184 [Int J Syst Evol 1958. Microbiol., 2018, 68: 3379–3393]. Type strain: DSM 43760 = JCM 14871 = ATCC 17929 = CCUG 20536 = CIP 101124 Schaalia georgiae (Johnson et al. 1990) = LMG 18080 = NCTC 9935. Nouioui et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces georgiae Johnson et al. Validation List no. 184 [Int J Syst Evol 1990. Microbiol., 2018, 68: 3379–3393]. Type strain: CCUG 32935 = DSM 6843 = ATCC 49285 = CIP 104749. Schaalia radingae (Wüst et al. 1995) Nouioui Reference: Front Microbiol., 2018, 9: 2007; et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Actinomyces radingae Wüst et al. Microbiol., 2018, 68: 3379–3393]. 1995 emend. Vandamme et al. 1998. Type strain: strain APL1 = CCUG 32394 = Schaalia hyovaginalis (Collins et al. 1993) DSM 9169 = ATCC 51856 = CCUG 34270 Nouioui et al. 2018, comb. nov. = LMG 15960. Basonym: Actinomyces hyovaginalis Collins et Reference: Front Microbiol., 2018, 9: 2007; al. 1993. Validation List no. 184 [Int J Syst Evol Type strain: strain BM 1192/5 = CCUG 35715 Microbiol., 2018, 68: 3379–3393]. = DSM 10695 = ATCC 51367 = CCUG 35604 = CIP 103923 = NCIMB 702983. Schaalia suimastitidis (Hoyles et al. 2001) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Actinomyces suimastitidis Hoyles et Microbiol., 2018, 68: 3379–3393]. al. 2001. Type strain: CCUG 39276 = CIP 106779. Schaalia meyeri (Cato et al. 1984) Nouioui et Reference: Front Microbiol., 2018, 9: 2007; al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Actinomyces meyeri (ex Prévot Microbiol., 2018, 68: 3379–3393]. 1938) Cato et al. 1984. Type strain: CCUG 21024 = DSM 20733 = Schaalia turicensis (Wüst et al. 1995) Nouioui ATCC 35568 = CIP 103148 = LMG 16161. et al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces turicensis Wüst et al. Validation List no. 184 [Int J Syst Evol 1995 Microbiol., 2018, 68: 3379–3393]. Type strain: strain APL10 = CCUG 32401 = DSM 9168 = ATCC 51857 = CCUG 34269 Schaalia naturae (Rao et al. 2012) Nouioui et = CIP 105357 = LMG 15961. al. 2018, comb. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Actinomyces naturae Rao et al. Validation List no. 184 [Int J Syst Evol 2012. Microbiol., 2018, 68: 3379–3393]. Type strain: strain BL-79 = CCUG 56698 = DSM 26713 = NRRL B-24670. Schaalia vaccimaxillae (Hall et al. 2003) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, comb. nov. Validation List no. 184 [Int J Syst Evol Basonym: Actinomyces vaccimaxillae Hall et Microbiol., 2018, 68: 3379–3393]. al. 2003. Type strain: strain R10176 = CCUG 46091 = Schaalia odontolytica (Batty 1958) Nouioui et CIP 107423.

S43 Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Microbiol., 2018, 68: 3379–3393]. Thermostaphylospora chromogena (McCarthy Thermomonospora amylolytica (Jiao et al. and Cross 1984) Wu et al. 2018, comb. nov. 2015) Nouioui et al. 2018, comb. nov. Basonym: Thermomonospora chromogena (ex Basonym: Actinomadura amylolytica Jiao et al. Krasil'nikov and Agre 1965) McCarthy and 2015. Cross 1984. Type strain: DSM 45822 = JCM 30324 = Type strain: strain 577 = ATCC 43196 = JCM CCTCC AA 201024. 6244 = DSM 43794 = NBRC 16096 = Reference: Front Microbiol., 2018, 9: 2007; NCIMB 10212 = NRRL B-16983. Validation List no. 184 [Int J Syst Evol Reference: Int J Syst Evol Microbiol., 2018, Microbiol., 2018, 68: 3379–3393]. 68: 602–608.

Thermomonospora cellulosilytica (Jiao et al. Winkia neuii (Funke et al. 1994) Nouioui et al. 2015) Nouioui et al. 2018, comb. nov. 2018, comb. nov. Basonym: Actinomadura cellulosilytica Jiao et Basonym: Actinomyces neuii Funke et al. 1994. al. 2015. Type strain: strain 97/90 = CCUG 32252 = Type strain: DSM 45823 = JCM 30326 = DSM 8576 = ATCC 51847 = CIP 104015. CCTCC AA 201023. Reference: Front Microbiol., 2018, 9: 2007; Reference: Front Microbiol., 2018, 9: 2007; Validation List no. 184 [Int J Syst Evol Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Microbiol., 2018, 68: 3379–3393]. Winkia neuii subsp. anitrata (Funke et al. Thermomonospora echinospora (Nonomura 1994) Nouioui et al. 2018, comb. nov. and Ohara 1971) Nouioui et al. 2018, comb. Basonym: Actinomyces neuii subsp. anitratus nov. Funke et al. 1994. Basonym: Microbispora echinospora Type strain: strain 50/90 = CCUG 32253 = Nonomura and Ohara 1971. DSM 8577 = ATCC 51849 = CIP 104016 = Type strain: DSM 43163 = JCM 3148 = ATCC LMG 14788. 27300 = BCRC 12547 = HUT 6548 = KCTC Reference: Front Microbiol., 2018, 9: 2007; 9313 = NBRC 14042. Validation List no. 184 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 2007; Microbiol., 2018, 68: 3379–3393]. Validation List no. 184 [Int J Syst Evol Microbiol., 2018, 68: 3379–3393]. Yinghuangia aomiensis (Nagai et al. 2011) Nouioui et al. 2018, comb. nov. Thermomonospora umbrina (Galatenko et al. Basonym: Streptomyces aomiensis Nagai et al. 1987) Nouioui et al. 2018, comb. nov. 2011. Basonym: Actinomadura umbrina Galatenko et Type strain: strain M24DS4 = DSM 42049 = al. 1987. NBRC 106164 = JCM 17986 = KACC Type strain: DSM 43927 = JCM 6837 = ATCC 14925. 49502 = INA 2309 = NBRC 14346 = NRRL Reference: Front Microbiol., 2018, 9: 2007; B-16244 = VKM Ac-1086. Validation List no. 184 [Int J Syst Evol Reference: Front Microbiol., 2018, 9: 2007; Microbiol., 2018, 68: 3379–3393].

NEW NOMENCLATURE

S44

Demequina maris (Hamada et al. 2015) NBRC 109395. Nouioui et al. 2018, nom. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Lysinimicrobium aestuarii Hamada Validation List no. 184 [Int J Syst Evol et al. 2015. Microbiol., 2018, 68: 3379–3393]. Type strain: strain HI12-104 = DSM 28144 = NBRC 109392. Micromonospora sediminimaris (Dai et al. Reference: Front Microbiol., 2018, 9: 2007; 2010) Nouioui et al. 2018, nom. nov. Validation List no. 184 [Int J Syst Evol Basonym: Verrucosispora sediminis Dai et al. Microbiol., 2018, 68: 3379–3393]. 2010. Type strain: strain MS426 = CGMCC 4.3550 = Demequina phytophila (Hamada et al. 2015) DSM 45558 = JCM 15670 =NBRC 107745. Nouioui et al. 2018, nom. nov. Reference: Front Microbiol., 2018, 9: 2007; Basonym: Lysinimicrobium flavum Hamada et Validation List no. 184 [Int J Syst Evol al. 2015. Microbiol., 2018, 68: 3379–3393]. Type strain: strain HI12-45 = DSM 28150 = NBRC 109391. Micromonospora trujilloniae (Xie et al. 2012) Reference: Front Microbiol., 2018, 9: 2007; Nouioui et al. 2018, nom. nov. Validation List no. 184 [Int J Syst Evol Basonym: Verrucosispora wenchangensis Xie Microbiol., 2018, 68: 3379–3393]. et al. 2012. Type strain: strain 234402 = CCTCC AA 20110 Demequina silvatica (Hamada et al. 2015) = DSM 45674 = JCM 31041 = NBRC Nouioui et al. 2018, nom. nov. 110790. Basonym: Lysinimicrobium luteum Hamada et Reference: Front Microbiol., 2018, 9: 2007; al. 2015. Validation List no. 184 [Int J Syst Evol Type strain: strain HI12-123 = DSM 28147 = Microbiol., 2018, 68: 3379–3393].

EMENDATION OF FAMILY

Mycobacteriaceae Chester 1897 emend. Gupta of changes in taxonomic opinion no. 28 [Int et al. 2018 J Syst Evol Microbiol., 2018, 68: 2137– Type genus: Mycobacterium Lehmann and 2138]. Neumann 1896. A member of the order Corynebacteriales. Reference: Front Microbiol., 2018, 9: 67; List

EMENDATION OF GENUS

Actinorectispora Quadri et al. 2016 emend. Actinotalea Yi et al. 2007 emend. Yan et al. Cao et al. 2018 2018 Type species: Actinorectispora indica Quadri et Type species: Actinotalea fermentans (Bagnara al. 2016. et al. 1985) Yi et al. 2007. Reference: Int J Syst Evol Microbiol., 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: 1023–1027. 68: 788–794. A member of the family Pseudonocardiaceae. A member of the family Cellulomonadaceae.

S45 Aestuariimicrobium Jung et al. 2007 emend. Jatrophihabitans Madhaiyan et al. 2013 Chen et al. 2018 emend. Lee et al. 2018 Type species: Aestuariimicrobium Type species: Jatrophihabitans endophyticus kwangyangense Jung et al. 2007. Madhaiyan et al. 2013 emend. Lee et al. Reference: Int J Syst Evol Microbiol., 2018, 2018. 68: 3296–3300. Reference: Int J Syst Evol Microbiol., 2018, A member of the family Propionibacteriaceae. 68: 1107–1111. A member of the family Frankiaceae. Geodermatophilus Luedemann 1968 emend. . Montero-Calasanz et al. 2017 Mycobacterium Lehmann and Neumann 1896 Type species: Geodermatophilus obscurus emend. Gupta et al. 2018 Luedemann 1968 emend. Montero-Calasanz Type species: Mycobacterium tuberculosis et al. 2017. (Zopf 1883) Lehmann and Neumann 1896. Reference: Front Microbiol., 2017, 8: 2501; Reference: Front Microbiol., 2018, 9: 57; List List of changes in taxonomic opinion no. 28 of changes in taxonomic opinion no. 28 [Int [Int J Syst Evol Microbiol., 2018, 68: 2137– J Syst Evol Microbiol., 2018, 68: 2137– 2138]. 2138]. A member of the family Geodermatophilaceae A member of the family Mycobacteriaceae.

EMENDATION OF SPECIES

Asaccharobacter celatus Minamida et al. 2008 Reference: Front Microbiol., 2017, 8: 2501; emend. Danylec et al. 2018 List of changes in taxonomic opinion no. 28 Type strain: strain do03 = AHU 1763 = DSM [Int J Syst Evol Microbiol., 2018, 68: 2137– 18785 = JCM 14811. 2138]. Reference: Int J Syst Evol Microbiol., 2018, 68: 1533–1540. Geodermatophilus aquaeductus Hezbri et al. 2015 emend. Montero-Calasanz et al. 2017 Geodermatophilus africanus Montero- Type strain: strain BMG801 = DSM 46834 = Calasanz et al. 2013 emend. Montero- CECT 8822. Calasanz et al. 2017 Reference: Front Microbiol., 2017, 8: 2501; Type strain: strain CF11/1 = DSM 45422 = List of changes in taxonomic opinion no. 28 CCUG 62969 = MTCC 11556. [Int J Syst Evol Microbiol., 2018, 68: 2137– Reference: Front Microbiol., 2017, 8: 2501; 2138]. List of changes in taxonomic opinion no. 28 [Int J Syst Evol Microbiol., 2018, 68: 2137– Geodermatophilus dictyosporus Montero- 2138]. Calasanz et al. 2015 emend. Montero- Calasanz et al. 2017 Geodermatophilus amargosae Montero- Type strain: strain G-5 = DSM 43161 = CCUG Calasanz et al. 2014 emend. Montero- 62970 = MTCC 11558 = ATCC 25080 = Calasanz et al. 2017 CBS 234.69 = KCC A-0154 = NBRC 13317. Type strain: strain G12 = strain G96 = DSM Reference: Front Microbiol., 2017, 8: 2501; 46136 = CCUG 62971 = MTCC 11559 = List of changes in taxonomic opinion no. 28 ATCC 25081 = JCM 3153 = NBRC 13316 = [Int J Syst Evol Microbiol., 2018, 68: 2137– NRRL B-3578 = KCTC 9360. 2138].

S46 2138]. Geodermatophilus nigrescens Nie et al. 2012 emend. Montero-Calasanz et al. 2017 Geodermatophilus ruber Zhang et al. 2011 Type strain: YIM 75980 = CCTCC AA emend. Montero-Calasanz et al. 2017 2011015 = JCM 18056. Type strain: CPCC 201356 = DSM 45317 = Reference: Front Microbiol., 2017, 8: 2501; CCM 7619. List of changes in taxonomic opinion no. 28 Reference: Front Microbiol., 2017, 8: 2501; [Int J Syst Evol Microbiol., 2018, 68: 2137– List of changes in taxonomic opinion no. 28 2138]. [Int J Syst Evol Microbiol., 2018, 68: 2137– 2138]. Geodermatophilus normandii Montero- Calasanz et al. 2013 emend. Montero- Geodermatophilus sabuli Hezbri et al. 2015 Calasanz et al. 2017 emend. Montero-Calasanz et al. 2017 Type strain: strain CF5/3 = DSM 45417 = Type strain: strain BMG 8133 = DSM 46844 = CCUG 62814 = MTCC 11412. CECT 8820. Reference: Front Microbiol., 2017, 8: 2501; Reference: Front Microbiol., 2017, 8: 2501; List of changes in taxonomic opinion no. 28 List of changes in taxonomic opinion no. 28 [Int J Syst Evol Microbiol., 2018, 68: 2137– [Int J Syst Evol Microbiol., 2018, 68: 2137– 2138]. 2138].

Geodermatophilus obscurus Luedemann 1968 Geodermatophilus saharensis Montero- emend. Montero-Calasanz et al. 2017 Calasanz et al. 2013 emend. Montero- Type strain: ATCC 25078 = DSM 43160 = Calasanz et al. 2017 NBRC 13315 = JCM 3152 = NRRL B-3577 Type strain: strain CF5/5 = DSM 45423 = = VKM Ac-658. CCUG 62813 = MTCC 11416. Reference: Front Microbiol., 2017, 8: 2501; Reference: Front Microbiol., 2017, 8: 2501; List of changes in taxonomic opinion no. 28 List of changes in taxonomic opinion no. 28 [Int J Syst Evol Microbiol., 2018, 68: 2137– [Int J Syst Evol Microbiol., 2018, 68: 2137– 2138]. 2138].

Geodermatophilus poikilotrophus corrig. Geodermatophilus siccatus Montero-Calasanz Montero-Calasanz et al. 2015 emend. et al. 2013 emend. Montero-Calasanz et al. Montero-Calasanz et al. 2017 2017 Type strain: strain G18 = DSM 44209 = CCUG Type strain: strain CF6/1 = DSM 45419 = 63018. CCUG 62765 = MTCC 11414. Reference: Front Microbiol., 2017, 8: 2501; Reference: Front Microbiol., 2017, 8: 2501; List of changes in taxonomic opinion no. 28 List of changes in taxonomic opinion no. 28 [Int J Syst Evol Microbiol., 2018, 68: 2137– [Int J Syst Evol Microbiol., 2018, 68: 2137– 2138]. 2138].

Geodermatophilus pulveris Hezbri et al. 2016 Geodermatophilus telluris Montero-Calasanz emend. Montero-Calasanz et al. 2017 et al. 2013 emend. Montero-Calasanz et al. Type strain: strain BMG 825 = CECT 9003 = 2017 DSM 46839. Type strain: strain CF9/1/1 = DSM 45421 = Reference: Front Microbiol., 2017, 8: 2501; CCUG 62764. List of changes in taxonomic opinion no. 28 Reference: Front Microbiol., 2017, 8: 2501; [Int J Syst Evol Microbiol., 2018, 68: 2137– List of changes in taxonomic opinion no. 28

S47 [Int J Syst Evol Microbiol., 2018, 68: 2137– Reference: Int J Syst Evol Microbiol., 2018, 2138]. 68: 1998–2005.

Jatrophihabitans endophyticus Madhaiyan et Mycobacterium tuberculosis (Zopf 1883) al. 2003 emend. Lee et al. 2018 Lehmann and Neumann 1896 emend. Riojas Type strain: strain S9-650 = DSM 45627 = et al. 2018 KACC 16232 = NBRC 109967. Type strain: strain H37Rv = ATCC 27294 = Reference: Int J Syst Evol Microbiol., 2018, NCTC 13114. 68: 1077–1111. Reference: Int J Syst Evol Microbiol., 2018, 68: 324–332. Jatrophihabitans fulvus Jin et al. 2015 emend. Lee et al. 2018 Streptomyces canus Heinemann et al. 1953 Type strain: strain PB158 = KCTC 33605 = emend. Kämpfer et al. 2018 JCM 30448. Type strain: strain BL 456786 = ATCC 12237 = Reference: Int J Syst Evol Microbiol., 2018, ATCC 19737 = BCRC 13652 = CBS 475.68 68: 1077–1111. = CGMCC 4.1468 = DSM 40017 = IFM 1092 = ISP 5017 = NBRC 12752 = JCM 4212 = Jatrophihabitans huperziae Kim et al. 2015 JCM 4569 = LMG 19329 = NCIMB 9627 = emend. Lee et al. 2018 NRRL B-1989 = NRRL B-3980 = NRRL-ISP Type strain: strain KIS75-12 = KACC 17298 = 5017 = RIA 1017 = UNIQEM 125 = VKM DSM 45908 = NBRC 109658. Ac-1011. Reference: Int J Syst Evol Microbiol., 2018, Reference: Int J Syst Evol Microbiol., 2018, 68: 1077–1111. 68: 42–46.

Lentzea soli Li et al. 2018 emend. Li et al. Streptomyces viridosporus Pridham et al. 1958 2018 emend. Goodfellow et al. 2017 Type strain: strain NEAU-LZC 7=CCTCC AA Type strain: ATCC 27479 = BCRC 11870 = 2017027=JCM 3238. CBS 654.72 = CCUG 37512 = DSM 40243 Reference: Int J Syst Evol Microbiol., 2018, = JCM 4859 = ISP 5243 = KCTC 9145 = 68: 3528–3533. NBRC 13353 = NCIMB 9824 = NRRL 2414 = NRRL ISP-5243 = RIA 1314 = VKM Ac- Mycobacterium intracellulare (Cuttino and 1769 = VKM Ac-618. McCabe 1949) Runyon 1965 emend. Reference: Antonie van Leeuwenhoek, 2017, Castejon et al. 2018 110: 1133–1148; List of changes in Type strain: ATCC 13950 = CCUG 28005 = taxonomic opinion no. 27 [Int J Syst Evol CIP 104243 = DSM 43223 = JCM 6384 = Microbiol., 2018, 68: 7–8]. NCTC 13025.

SYNONYM

Mycobacterium africanum Castets et al. 1969 pro synon. Mycobacterium tuberculosis Mycobacterium bovis Karlson and Lessel 1970 (Zopf 1883) Lehmann and Neumann 1896 pro synon. Mycobacterium tuberculosis emend. Riojas et al. 2018 (Zopf 1883) Lehmann and Neumann 1896 Reference: Int. J. Syst. Evol. Microbiol., 2018, emend. Riojas et al. 2018 68: 324–332. Reference: Int. J. Syst. Evol. Microbiol., 2018,

S48 68: 324–332. 68: 324–332.

Mycobacterium caprae (Aranaz et al. 1999) Mycobacterium pinnipedii Cousins et al. 2003 Aranaz et al. 2003 pro synon. pro synon. Mycobacterium tuberculosis Mycobacterium tuberculosis (Zopf 1883) (Zopf 1883) Lehmann and Neumann 1896 Lehmann and Neumann 1896 emend. Riojas emend. Riojas et al. 2018 et al. 2018 Reference: Int. J. Syst. Evol. Microbiol., 2018, Reference: Int. J. Syst. Evol. Microbiol., 2018, 68: 324–332. 68: 324–332. Streptomyces ciscaucasicus Sveshnikova et al. Mycobacterium microti Reed 1957 pro synon. 1986 pro synon. Streptomyces canus Mycobacterium tuberculosis (Zopf 1883) Heinemann 1953 Lehmann and Neumann 1896 emend. Riojas Reference: Int. J. Syst. Evol. Microbiol., 2018, et al. 2018 68: 42–46. Reference: Int. J. Syst. Evol. Microbiol., 2018,

S49 Ōmura award 2018

Investigation of secondary metabolite diversity in actinomycetes using spectrochemical approach

Yasuhiro Igarashi

Biotechnology Research Center, Toyama Prefectural University

INTRODUCTION of compounds regarding to the variety of substituents and ring patterns and the abundant chiral centers was expected. It was around the year 1996 when I started the screening of Accordingly, compounds possessing the UV absorption bioactive compounds from natural resources. At that time, maxima at approximately shorter than 300 nm were chosen, pharmaceutical companies were getting less interested in since many of the compounds produced by type I PKS pathway natural products in their drug discovery scheme, due to the have a relatively short-conjugated system in their structures. increasing frequency of re-isolation of known compounds and Compounds showing the UV spectra for the benzene or indole the decreasing frequency of discovering new compounds. ring, and the end-absorption were excluded from the candidate Researchers were suspicious about the potency of as they were considered as simple peptides. Pigmented actinomycetes as a producer of new bioactive compounds. On compounds with the UV absorption at over 400 nm were also the other hand, since the previous screening from discarded since such pigments had been studied for many years microorganisms in industry or academia had been conducted in owing to the easy detectability. an activity-guided manner, careful attention had not been paid Based on these considerations, spectroscopic screening was to the compounds which did not show the target activity begun to explore the structural diversity and biosynthetic although a number of such “not-active” compounds were capability of secondary metabolites in actinomycetes. present in the screening extracts. In addition, the genomic information of actinomycetes was not yet available regarding to 1. APPLICATION OF UV SPECTROSCOPIC the secondary metabolite genes. Meanwhile, in my own ANALYSIS TO EXPLORE THE SECONDARY experiences in analyzing HPLC data, culture extracts often METABOLISM IN A SINGLE STRAIN contained unknown peaks in addition to the target active principles, which led to the idea of comprehensive investigation To validate the applicability of this methodology, one strain of the products of actinomycetes at the component level to of Streptomyces was selected for the exhaustive analysis of elucidate the diversity of secondary metabolism in secondary metabolites as an initial attempt. The strain S. actinomycetes. In the field of pharmacognosy, it is often hygroscopicus TP-A0449 was isolated from the stem of bracken observed that the activity is derived not only from a single (Pteridium aquilinum) collected in Toyama, Japan. This strain compound but from multiple components, which facilitated the was producing diverse metabolites of high polarity to low exhaustive analysis of compounds regardless of the biological polarity. Azalomycin B (elaiophylin) was the most major activity. This kind of “exhaustive” approach seemed suitable to product and some additional compounds such as ansamycins of disclose the biosynthetic capacity of the secondary metabolism herbimycin/geldanamycin-class and 6-prenylindole were in actinomycetes. produced. Afterwards, I noticed that these three types of UV spectrum is currently the most common, the first-choice secondary metabolites were often associated with other S. physico-chemical property for the selection of “probably new” hygroscopicus strains, and proposed to call them as “marker compounds in the crude extract of natural sources. In our metabolites” for S. hygroscopicus. Detailed analysis of the laboratory, the in-house HPLC-UV database was used to products from strain TP-A0449 led to the finding of three new eliminate known compounds from hit samples but it had not polyketides derived from type I PKS, pteridic acids A and B been positively employed for chemical screening. UV spectrum [Igarashi, 2002] and pterocidin [Igarashi, 2006; Igarashi, not only provides the structural information of the conjugated 2012a]. Pteridic acid A is the plant hormone-like growth (unsaturated) system but also reflects the biosynthetic trait of activator which induces adventitious root formation at 0.001 to the compounds. It was assumed that UV spectroscopic analysis 0.1 M in green beans. It also promotes the proliferation of could be also used to select compounds derived from a given, tobacco BY2 cells at 0.1 to 3 M. Pterocidin shows anti- specific biosynthetic pathway. In the early stage of this research, invasion activity against murine colon 26-L5 carcinoma cells high priority for structural analysis was given to type I PKS with an IC50 of 0.25 M. products because the higher structural diversity than other types

S50 A254 nm azalomycin B TAN420A hygrocin B and and congeners and congeners congeners

pteridic acid A pteridicacid B 6-prenylindole galbonolide A galbonolide B

pterocidin

Fig. 1. Exhaustive analysis of secondary metabolites produced by Streptomyces hygroscopicus TP-A0451.

2. DISCOVERY OF BIOSYNTHETICALLY membered tetronic acid-like ring system, 2H-tetrahydro-4,6- UNIQUE SECONDARY METABOLITES BY dioxo-1,2-oxazine (TDO). As this ring system was quite SPECTROSCOPIC SCREENING unusual in natural products, it became necessary to prove this Prompted by the promising results from the single-strain structure through more robust method. Fortunately, crystals analysis described above, our spectrochemical approach was suitable to X-ray diffraction analysis were obtained and the expanded to actinomycetes of various origins in our screening absolute configuration of alchivemycin A as well as the TDO program. In the early 2000s, we were focusing on the ring structure was unambiguously determined, in combination actinomycetes of plant-origin and tropical areas as an unstudied or under-evaluated source, expecting to obtain structurally interesting molecules from such unutilized niches.

1) Alchivemycin Alchivemycins A and B are the polyketides found from the culture extract of Streptomyces sp. TP-A0867 collected from a leaf of chive (Allium tuberosum) [Igarashi, 2010a; Kim, 2013]. The UV spectra of alchivemycins showed an absorption pattern typical for tetronic acid (max 222, 282 nm). The planar structure of alchivemycin A was initially proposed on the basis of NMR Fig. 3. Biosynthetic origin of alchivemycin A. spectroscopic data of alchivemycin A and its pentaacetate. Our analytical data suggested the presence of an unprecedented six- with the chiral anisotropic analysis. The biosynthetic origin of the TDO unit was studied by using the stable isotope labeling experiment and bioinformatics analysis of the biosynthetic genes. Acetate and propionate were incorporated into the polyketide backbone, whereas one carbon directly connecting the nitrogen atom in the TDO ring was labeled by 1-13C-glycine and N-hydroxy-1-13C-glycine. The in silico analysis of the gene cluster indicated the presence of one NRPS gene of which the adenylation domain possessed the signature sequence for glycine incorporation. Based on these evidences, we proposed the biosynthetic pathway for alchivemycins in which the TDO portion was assembled by the condensation of N- Fig. 2. Structures of alchivemycins A and B. hydroxyglycine derived from glycine to the polyketide chain [Komaki, 2016a]. Both alchivemycins A and B displayed potent

S51 antimicrobial activity against Micrococcus luteus in ng/mL ranges and inhibited tumor cell invasion at micromolar to 3) Jomthonic acids and antarlides submicromolar concentrations. No additional natural products Streptomyces sp. BB47 was isolated from a soil sample related to alchivemycin have been reported to date. collected in Thailand. Strain BB47 was found to produce two known classes of compounds, piericidins (a linear polyketide 2) Nonthmicin and campechic acid with a pyridinone ring) and clethramycin (a linear hexaene Nonthmicin and ecteinamycin were isolated from a culture polyketide bearing a sulfonate functionality), and jomthonic extract of Actinomadura strain K4S16 which was obtained from acids as a new class of secondary metabolites. Jomthonic acids the rhizosphere soil of a rice field in Thailand [Igarashi, 2017]. are the small molecules which consist of an unsaturated fatty Both compounds displayed UV absorption maxima at 227 and acid, -methylphenylalanine, and -hydroxybutanoic acid and 298 nm, suggestive of the presence of tetronic acid-like show UV absorption at 264 nm [Igarashi, 2012b; Yu, 2014b]. functionality. Planar structures of these compounds were -Methylphenylalanine is a very rare unusual amino acid in elucidated through careful examination of NMR and MS data. nature. The (2S,3S)-isomer was found in the metabolites of At the moment we assigned the structures, both compounds Streptomyces (bottromycin) and Alternaria (AK-toxin), but the were not reported anywhere, however later ecteinamycin was (2S,3R)-isomer in jomthonic acids is the first case in natural found to be filed in a patent by the research group of the US. products. Jomthonic acids induced differentiation of murine Ecteinamycin is a polyether polyketide isolated from a marine- preadipocyte ST-13 cells into the matured adipocyte cells at 2 derived Actinomadura. In order to establish the configuration to 50 M. of multiple chiral centers, crystals of ecteinamycin were subjected to the X-ray crystallographic analysis, which defined the absolute configuration and its coordination to a sodium cation through six oxygen functionalities. The absolute configuration of nonthmicin was proposed to be the same as ecteinamycin in consideration of biosynthetic relationship. The Fig. 5. Structures of jomthonic acids. tetronic acid moiety substituted with an exo-methylene group is not an uncommon structural unit in microbial metabolites, but After the discovery of jomthonic acids, strain BB47 drew nonthmicin is the first example in which the tetronic acid part our attention again for its inhibitory activity against the binding is modified by a halogen atom. Draft genome analysis of strain of dihydroxytestosterone to the androgen receptor. Careful K4S16 allowed the tentative assignment of the biosynthetic inspection of the metabolites in the crude extract revealed the gene cluster for nonthmicin/ecteinamicin and the identification production of additional, unknown metabolites displaying a of a putative halogenase gene responsible for the halogenation broad UV absorption around 340 nm, implying a polyolefinic of the tetronic acid moiety. Supplementation of sodium bromide functionality. Activity-guided purification resulted in the into the culture actually gave the production of a bromo isolation of a series of novel polyene-type macrolides, analogue of nonthmicin. Nonthimicin exhibited various antarlides [Saito, 2016; Saito, 2017]. The most striking feature biological activity: very potent antimicrobial activity against of antarlides is the m-hydroxyphenyl starter unit for PKS Gram-positive bacteria with MIC of 0.001 to 0.005 g/mL; biosynthesis. Among the various type I PKS starters, benzene autophagy inducing activity at 0.01 to 0.1 M; inhibition of type is quite uncommon: soraphen A, a macrolide of Sorangium tumor cell invasion with an IC50 of 0.015 M. (myxobacteria) has a phenyl starter and aplysiatoxin, a macrolide of Lyngbya (cyanobacteria) possesses a 2-bromo-4- hydroxyphenyl starter. These benzene-type starters are derived from the shikimate pathway. In the secondary metabolites of

Fig. 4. Structures of nonthimicin and campechic acids.

Campechic acids are the polyether-like linear polyketides produced by a Streptomyces strain CHI93 isolated from a rock Fig. 6. Structures of antarlides. sample in Mexico [Yu, 2014a]. The absolute configuration was determined by the combination of chemical degradation and actinomycetes, the starter unit of FK506 is also derived from chiral anisotropic analyses and the total synthesis [Isaka, 2016]. shikimate but it is an oxygenated cyclohexane starter. Antarlide Similar to nonthmicin, campechic acids, specifically campechic is the first example of type I PKS products which have the acid A, showed very potent inhibitory activity toward the tumor benzene type starter within actinomycete metabolites. This cell invasion at nanomolar concentrations.

S52 structurally unique macrolide potently inhibits the binding are encoded adjacent to the PKS gene cluster for the linfuranone between dihydroxytestosterone and the androgen receptor at a biosynthesis. Two are assigned to catalyze the oxidative comparable level to the antitumor drug flutamide in a formation of furanone functionality from the nascent PKS micromolar range. product and one is proposed to catalyze Baeyer-Villiger oxidation of the carbon chain of linfuranone C to yield 4) Lifuranones linfuranone B. Baeyer-Villiger oxidation is known to be 3(2H)-Furanone is a relatively minor functionality in natural involved in the biosynthesis of natural products, but oxidative products. To date, about 20 polyketides possessing the furanone cleavage of a linear carbon chain is uncommon. Linfuranones functionality are known from bacteria, fungi, and marine were shown to induce the preadipocyte differentiation into the invertebrates. Linfuranones are the new member of furanone- matured adipocytes at 2 to 50 M. Linfuranone B also inhibited containing polyketides isolated from the culture extract of an the tumor cell invasion at 2.1 M. endophytic Sphaerimonospora strain GMKU363 collected in Thailand. Initially, we isolated two new polyketides, 5) Other new compounds More diverse secondary metabolites were obtained through the UV-based chemical screening, ranging from polyketides to amino acid-, carbohydrate-, and nucleic acid-derivatives [Igarashi, 2011a; Igarashi, 2007; Igarashi, 2011b; Igarashi, 2012c; Igarashi, 2017b; Igarashi, 2012d; Kim, 2014; Igarashi, 2009; Igarashi, 2010b; Oku, 2014; Komaki, 2016b].

CONCLUDING REMARKS

Initially, our chemical sreening employing the UV spectral database was applied to the exhaustive analysis of secondary metabolites in a single strain to validate the biosynthetic capacity of one strain. Careful analysis of a Streptomyces strain resulted in the discovery of new polyketides, pteridic acids with Fig. 7. Structures of linfuraones. plant-growth promoting activity and pterocidin with antiinvasive activity. This methodology was further applied to the linfuranone A [Indananda, 2013] and its acyl congener screening from actinomycetes of plant or tropical zone origin, linfuranone B [Akiyama, 2018]. Structures of these compounds in consideration of the uniqueness of collection sites, leading to suggested that linfuranone B was produced from linfuranone A the discovery of a series of biosynthetically unique new by acylation. However, the in silico analysis of PKS gene compounds including alchivemycin, nonthimicin, jomthonic function for linfuranone biosynthesis indicated that the actual acid, antarlide, and brartemicin bearing various bioactivities. PKS product could have a polyketide carbon chain five-carbon Through this research, the effectiveness of our spcetrochemical longer than linfuranones A and B [Komaki, 2015]. Then, we approach in natural product screening was proven. reexamined the culture condition for strain GMKU363, and When this research was initiated in the late 1990s, chemical found that this strain grew faster at slightly higher temperature screening or spectroscopic screening was not so much common around 37°C than around 30°C and produced higher amount of in natural product screening from microorganisms. In recent linfuranone B and some additional new metabolites possessing years, however, many of the papers reporting new microbial the same UV spectral pattern as linfuranones. The most secondary metabolites are likely employing the structure- abundant peak was then isolated and identified as a new oriented chemical screening using LC/UV or LC/UV/MS to congener, linfuranone C which possessed the carbon skeleton discover new skeletons. A number of researchers are continuing predicted by the annotation of the PKS genes. Three oxygenases to pursue the new chemical structures, and the biosynthetic

Fig. 8. Structures of new bioactive compounds discovered by UV spectroscopic screening.

S53 capacity of actinomycetes is being disclosed deeper and wider Igarashi, Y., et al. (2012a). Absolute configuration of than expected 20 years ago. Continuous survey on the pterocidin, a potent inhibitor of tumor cell invasion from a actinomycetes of unstudied niches or the unstudied rare species marine-derived Streptomyces. Tetrahedron Lett. 53, 654- will uncover the metabolic potential of actinomycetes for future 656. biotechnology. Igarashi, Y., et al. (2012b). Jomthonic acid A, a modified amino acid from a soil-derived Streptomyces. J. Nat. Prod. ACKNOWELDGEMENTS 75, 986-990. Igarashi, Y., et al. (2012c). Prajinamide, a new modified This research was conducted in the Laboratory of peptide from a soil-derived Streptomyces. J. Antibiot. 65, Microbial Engineering (former name: Laboratory of 157-159. Exploratory Biotechnology) at Toyama Prefectural University, Igarashi, Y., et al. (2012d). Catechoserine, a new catecholate- Japan. The author expresses special thanks to the late Professor type inhibitor of tumor cell invasion from Streptomyces sp. Toshikazu Oki, the Professor Tamotsu Furumai, the students J. Antibiot. 65, 207-209. and graduates from this laboratory, and the collaborators in and Igarashi, Y., et al. (2017). Nonthmicin, a polyether polyketide out of Japan. The author also expresses his gratitude to all of the bearing a halogen-modified tetronate with neuroprotective pioneering researchers in the Society for Actinomycetes Japan and antiinvasive activity from Actinomadura sp. Org. Lett. who always inspired him to continue this research. 19, 1406-1409. This review article is dedicated to the late Professors Indananda, C., et al. (2013). Linfuranone A, a new polyketide Toshikazu Oki (1935.1–2019.4, Toyama Prefectural from plant-derived Microbispora sp. GMKU363. J. University), Takuya Nihira (1953.1–2018.9, Osaka University), Antibiot. 66, 675-677. and Kenji Mori (1935.3–2019.4, University of Tokyo) who Isaka R., et al. (2016). Complete stereochemical assignment of gave an opportunity to the author to study natural product campechic acids A and B. J. Org. Chem. 81, 3638-3647. chemistry and actinomycetology. Kim, Y., et al. (2013). Biosynthetic origin of alchivemycin A, a new polyketide from Streptomyces and absolute REFERENCES configuration of alchivemycin B. Org. Lett. 15, 3514-3517. Kim, Y., et al. (2014). Nocapyrones: - and -pyrones from a Akiyama, H., et al. (2018). Linfuranones B and C, furanone- marine-derived Nocardiopsis sp. Mar. Drugs 12, 4110- containing polyketides from a plant-associated 4125. Sphaerimonospora mesophila. J. Nat. Prod. 81, 1561-1569. Komaki, H., et al. (2015). Draft genome sequence of Igarashi, Y., et al. (2002). Pteridic acids A and B, novel plant linfuranone producer Microbispora sp. GMKU363. growth promoters with auxin-like activity from Genome Annouc. 3, e01474-15. Streptomyces hygroscopicus TP-A0451. J. Antibiot. 55, Komaki, H., et al. (2016a). Draft genome sequence of 764-767. Streptomyces sp. TP-A0867, an alchivemycin producer. Igarashi, Y., et al. (2006). Pterocidin, a cytotoxic compound Stand. Genomic Sci. 11, 85. from the endophytic Streptomyces hygroscopicus. J. Komaki, H., et al. (2016b). Draft genome sequence of Antibiot. 59, 193-195. Streptomyces sp. MWW064 for elucidating the rakicidin Igarashi, Y., et al. (2007). Antitumor anthraquinones from an biosynthetic pathway. Stand. Genomic Sci. 11, 83. endophytic actinomycete Micromonospora lupini sp. nov Oku, N., et al. (2014). Complete stereochemistry and Bioorg. Med. Chem. Lett. 17, 3702-3705. preliminary structure-activity relationship of rakicidin A, a Igarashi, Y., et al. (2009). Brartemicin, an inhibitor of tumor hypoxia-selective cytotoxin from Micromonospora spp. J. cell invasion from the actinomycete Nonomuraea sp. J. Nat. Nat. Prod. 77, 2561-2565. Prod. 72, 980-982. Saito, S., et al. (2016). Antarlides: a new type of androgen Igarashi, Y., et al. (2010a). Alchivemycin A, a bioactive rectptor (AR) antagonist that overcomes resistance to AR- polycyclic polyketide with an unprecedented skeleton from targeted therapy. Angew. Chem. Int. Ed. 55, 2728-2732. Streptomyces sp. Org. Lett. 12, 3402-3405. Saito, S., et al. (2017). Antarlides F-H, new members of the Igarashi, Y., et al. (2010b). Rakicidin D, an inhibitor of tumor antarlide family produced by Streptomyces sp. BB47. J. cell invasion from marine-derived Streptomyces sp. J. Antibiot. 70, 595-600. Antibiot. 63, 563-565. Yu, L., et al. (2014a). Campechic acids A and B; anti-invasive Igarashi, Y., et al. (2011a). Maklamicin, an antibacterial polyether polyketides from a soil-derived Streptomyces. J. polyketide from an endophytic Micromonospora sp. J. Nat. Nat. Prod. 77, 976-982. Prod. 74, 670-674. Yu, L., et al. (2014b). Jomthonic acids B and C, two new Igarashi, Y., et al. (2011b). Lupinacidin C, an inhibitor of modified amino acids from Streptomyces sp. J. Antibiot. 67, tumor cell invasion from Micromonospora lunipi J. Nat. 345-347. Prod. 74, 862-865.

S54

Publication of Award Lecture

The Society for Actinomycetes Japan Ōmura Award 2018,

Dr. Yasuhiro Igarashi

“Investigation of secondary metabolite diversity in actinomycetes using spectrochemical approach” Actimomycetologica (2019) 33 [1], S50-S54.

Biotechnology Research Center, Toyama Prefectural University

S55 Hamada award 2017 Isolation of endophytic actinomycetes and the search for new secondary metabolites

Yuki Inahashi

Kitasato Institute for Life Sciences, Kitasato University

INTRODUCTION compounds from secondary metabolites of the isolates, and their biosynthesis. The Actinomycetes are a significantly unique and important source of bioactive compounds. These include 1. Isolation of actinomycetes from plant samples streptomycin (used to treat bacterial diseases including There is no universally accepted definition of a bacterial tuberculosis) and tacrolimus, also known as FK506, (an “endophyte”. For the purposes of this report, that of Hallmann immunosuppressive drug). In addition, the avermectins are et al. (1997) will be used, namely that an endophyte is any unprecedented endectocides, which are boosting livestock bacterium that can be isolated from surface-disinfected plant production, killing crop pests and helping to eliminate two of tissue, or extracted from inside the plant, and that the organism the world’s most devastating tropical diseases, onchocerciasis lives symbiotically with the plant or does not harm the plant. and lymphatic filariasis. However, over the past several Endophytic actinomycetes were isolated from the roots, stems decades, it has become increasingly more difficult to find new and leaves of plants after removal of soil using tap water useful bioactive compounds from secondary metabolites of followed by drying in a chamber with silica gel. The surface of actinomycetes (Berdy, 2012). Soil is the dominant habitat of all plant samples was then sterilized with 70% ethanol these microorganisms and many actinomycetes have been followed by 1% sodium hypochlorite. The samples were then isolated from soil samples. Clearly, it is advantageous to rinsed in sterilized water and ground with a mortar and pestle explore new strains from hitherto untapped sources in order to in an extraction buffer [0.38% K2HPO4, 0.12% KH2PO4, discover novel compounds. Plants are also a source of 0.51% MgSO4·7H2O, 0.25% NaCl, 0.005% Fe2(SO4)3·nH2O, actinomycetes. There have been several reports of endophytic 0.005% MnSO4·H2O]. The resultant liquid was diluted with actinomycetes, their metabolites and symbiotic interactions same buffer and mixed into cellobiose asparagine agar (1.0%

(Igarashi et al., 2004; Hasegawa et al., 2006; Qin et al., 2011; cellobiose, 0.1% L-asparagine, 0.1% K2HPO4, 0.0001% T Matsumoto et al., 2017). Micromonospora lupini Lupac 08 FeSO4·7H2O, 0.0001% MnCl2·4H2O, 0.0001% ZnSO4. 7H2O, and M. saelicesensis Lupac 09T are new species isolated from 1.5% agar, pH 7.0) and water proline agar (1.0% proline, 1.5% root nodules of Lupinus angustifolius (Trujillo et al., 2007) agar, tap water, pH not adjusted), which contained nalidixic and the strain Lupac 08T has plant-growth promoting ability acid (25 μg mL-1) and benomyl (20 μg mL-1). Actinomycete (Benito et al., 2017). Igarashi et al. found a plant-growth colonies were picked up after incubation for 1-4 weeks at activator, pteridic acid A, from the metabolites of 27°C. For comparative purposes, actinomycetes were also Streptomyces hygroscopicus TP-A0451 isolated from isolated from rhizospheric soils. The genera of isolates were Pteridium aquilinum (Igarashi et al., 2002). Munumbicins, identified by BLAST search of 16S rRNA gene partial which display broad-spectrum activities against pathogenic sequences. Classification of actinomycetes isolated from plant fungi and bacteria, were discovered from the metabolites of and soil samples is shown in Fig. 1. One thousand and Streptomyces sp. NRRL 30562, isolated from a medicinal thirty-four strains isolated from 29 plant samples were plant, Kennedia nigriscans (Castillo et al., 2012). More than 1 taxonomically grouped into 29 genera [Streptomyces (272 million plants, including 350,000 known species, 470,000 strains, 26.0%), Micromonospora (241 strains, 23.0%), synonyms and 242,000 unresolved species have been reported Dactylosporangium (89 strains, 8.6%), Microbispora (75 (The Plant List, 2013). The biodiversity of endophytic strains, 7.3%), Nonomuraea (60 strains, 5.8%), actinomycetes existing under different host plants, or living Sphaerisporangium (50 strains, 5.0%), Actinoallomurus (48 symbiotically with specific plants, has yet to be properly strains, 4.6%), Planotetraspora (46 strains, 4.4%), explored and it seems likely this will reveal a cornucopia of Acrocarpospora (27 strains, 2.6%), Polymorphospora (23 new actinomycetes and novel compounds. strains, 2.2%), Agromyces (20 strains, 1.9%), Phytohabitans Herein, I describe results from our research work with (14 strains, 1.4%), Verrucosispora (12 strains, 1.2%), respect to isolation of actinomycetes from plants, including the Amycolatopsis (10 strains, 1.0%), Streptosporangium (9 taxonomy of actinomycete strains isolated, new bioactive strains, 0.9%), Asanoa (8 strains, 0.8%), Actinophytocola (6

S56 Fig. 1. Classification of actinomycetes isolated from plant and soil samples.

strains, 0.6%), Nocardia (6 strains, 0.6%), Actinomadura (4 further taxonomic studies (for details, see section). Thus, strains, 0.4%), Quadrisphaera (2 strains, 0.2%), plants represent a potentially productive source of rare Plantactinospora (2 strains, 0.2%), Actinaurispora (2 strains, actinomycetes, including novel taxonomic groups. The most 0.2%), Actinoplanes (2 strains, 0.2%), Actinocorallia (1 strain, predominant strains isolated from plants belonging to the 0.1%), Microtetraspora (1 strain, 0.1%), Planomonospora (1 family Micromonosporaceae (Micromonospora, strain, 0.1%), Herbidospora (1 strain, 0.1%), Dactylosporangium, Polymorphospora, Phytohabitans, Kibdelosporangium (1 strain, 0.1%) and Kitasatospora (1 Verrucosispora, Asanoa, Plantactinospora and Actinoplanes). strain, 0.1%)]. Four hundred and thirty-six strains isolated To date, the family Micromonosporaceae consists of 29 genera from 6 rhizospheric soil samples were taxonomically grouped and 6 of those, Phytohabitans (Inahashi et al., 2010), into 19 genera [Streptomyces (351 strains, 81.0%), Rhizocola (Matsumoto et al., 2014), Plantactinospora (Qin et Kitasatospora (22 strains, 5.1%), Lentzea (16 strains, 3.7%), al., 2009), Phytomonospora (Li et al., 2011), Kribbella (9 strains, 2.1%), Nonomuraea (7 strains, 1.6%), Actinorhabdospora (Mingma et al., 2016) and Kibderosporangium (6 strains, 1.4%), Amycolatopsis (4 strains, Mangrovihabitans (Liu et al., 2017) were discovered from 0.9%), Nocardia (4 strains, 0.9%), Dactylosporangium (3 plants. Trujillo et al. have also reported that the genus strains, 0.7%), Micromonospora (3 strains, 0.7%), Micromonospora is widespread in legume nodules, the strains Actinomadura (2 strains, 0.5%), Catellatospora (1 strain, contributing to plant development and health by acting as 0.2%), Actinoplanes (1 strain, 0.2%), Herbidospora (1 strain, plant growth-promoting bacteria (Martinez-Hidalgo et al., 0.2%), Kutzneria (1 strain, 0.2%), Microbispora (1 strain, 2014; Trujillo et al., 2015; Benito et al., 2017). Plants appear 0.2%), Pseudonocardia (1 strain, 0.2%), Saccharothrix (1 to be suitable habitats for strains of the Micromonosporaceae strain, 0.2%), Streptosporangium (1 strain, 0.2%) and and it is likely that symbiotic relationships have evolved Umezawaea (1 strain, 0.2%)]. As it is well known that soils are between the plants and those bacteria. a rich source of Streptomyces, unsurprisingly, most strains isolated from the rhizospheric soils were Streptomyces. 2. Taxonomic study of novel endophytic actinomycetes Conversely, >70% of strains isolated from plants were 2-1. Phytohabitans suffuscus gen. nov., sp. nov., P. flavus sp. non-Streptomyces, and proved to be rare actinomycetes, those nov., P. rumicis sp. nov. and P. houttuyneae sp. nov. in the strains belonging to various genera. Furthermore, a novel family Micromonosporaceae genus, Phytohabitans, containing 4 novel species (P. suffuscus, Actinomycete strains K07-0523T, K09-0627T, K11-0047T P. flavus, P. rumicis, P. houttuyneae) along with a new species, and K11-0057T (Fig. 2) were isolated from roots of Goodyera Streptoporangium oxazolinicum, were proposed following procera, Rumex acetosa and Houttuynia cordata, respectively.

S57 Fig. 2. Scanning electron micrographs of isolates.

Fig. 3. Neighbor-joining tree based on 16S rDNA sequences Fig. 4. Neighbor-joining tree based on 16S rDNA sequences showing relationship between Phytohabitans species and related showing relationship between strain K07-0460T and related actinomycetes. The numbers at nodes indicate bootstrap values actinomycetes. The numbers at nodes indicate bootstrap values based on an analysis of 1000 re-sampled datasets, only values based on an analysis of 1000 re-sampled datasets, only values above 50% are given. Bar, 0.01 nucleotide substitutions per site. above 50% are given. Bar, 0.01 nucleotide substitutions per site.

They were classified within the Micromonosporaceae by acid type of the strains were negative, type II and 2d, phylogenetic analysis based on 16S rRNA gene sequences (Fig. respectively, and the characteristics were different from those 3). The phylogenetic analysis indicated that the strains were of the genus Catenuloplanes (Yokota et al., 1993). related to the genera Asanoa and Catenuloplanes. The strains Furthermore, the unique characteristic, namely the cell wall contained galactose and xylose as diagnostic sugars, while the containing both meso-diaminopimelic acid and L-lysine as genus Asanoa contains arabinose, galactose and xylose (Lee & Hah, 2002). The spore motility, phospholipid type and fatty

S58 Fig. 5. Structures of spoxazomicins A-C, actinoallolides A-E and trehangelins A-C.

diamino acid, distinguished the strains from other genera of (C17:0 10-methyl, C17:18c and C17:0), and MK-9 (H2) and the family Micromonosporacea. Based on the phylogenetical, MK-9 (H4) as predominant menaquinones. The phylogenetic morphological and chemotaxonomical properties, a new genus analysis, based on 16 rRNA gene sequences, indicated that the Phytohabitans (Phy.to.ha’bi.tans. Gr.n. phyton plant; L. part. strain was clustered within the genus Streptosporangium (Fig. adj. habitans inhabiting; N. L. part. adj. used as a masc. n. 4). The morphological, chemotaxonomical and phylogenetical Phytohabitans plant-inhabiting) was proposed (Inahashi et al., properties revealed that the strain belonged to the genus 2010). The type species and type strain is P. suffuscus Streptosporangium. Furthermore, DNA-DNA relatedness (suf.fus’cus. L. masc. adj. suffuscus brownish) K07-0523T. As values with strain K07-0460T among the related strains, S. the result of DNA-DNA hybridization, the relatedness values amethystogenes subsp. amethystogenes DSM43179T and S. among the strains K07-0523T, K09-0627T, K11-0047T and amethystogenes subsp. fukuiense IFO 15365T, were below K11-0057T are below 70%. Thus, 3 new species, P. flavus 70%. Based on the above results, strain K07-0460T (fla’vus. L. masc. adj. flavus yellow), P. rumicis (ru’mi.cis. L. represented a new species of the genus Streptosporangium, for n. rumex -icis sorrel and also a scientific genus name; L. gen. which the name S. oxazolinicum (o.xa.zo.li’nicum. N.L. n. n. rumicis of sorrel), P. houttuyneae (hout.tuy.ne’ae. N.L. gen. oxazolinum, oxaoline; L. neut. suff. –icum, suffix used with n. houttuyneae of Houttuynea) were proposed (Inahashi et al., the sense of pertaining to; N.L. neut. adj. oxazolinicum, 2010; Inahashi et al., 2012). The type strains of P. flavus, P. pertaining to oxazoline, referring to the production of rumicis and P. houttuyneae are K09-0627T, K11-0047T and oxazoline compounds) was proposed (Inahashi et al., 2011a). K11-0057T, respectively. 3. Search for new bioactive compounds from secondary 2-2. Streptosporangium oxazolinicum sp. nov. metabolites of isolates and their biosynthesis An actinomycete strain K07-0460T, isolated from roots of 3-1. Spoxazomicin Goodyera procera, forms globose sporangia on aerial mycelia Several useful microbial metabolites, such as (Fig. 2). Whole-cell sugars are glucose, galactose, mannose, staurosporine, dityromycin and pyrindicin, have been madurose, ribose and xylose. Diagnostic diamino acid, the discovered by alkaloid screening using Dragendroff’s reagent acyl type of muramic acid, phospholipid type and mycolic acid (Ōmura et al., 1974; Ōmura et al., 1977a; Ōmura et al., 1977b). are meso-diaminopimeric acid, N-acetyl, type IV and absent, In the course of screening culture broths of endophytic respectively. The cells contain major amounts of actinomycetes, three new alkaloid antibiotics, spoxazomicins 10-methyl-branched, saturated and unsaturated fatty acids A, B and C (Fig. 5) (Inahashi et al., 2011b), were discovered

S59 Fig. 6. The gene cluster and proposed pathway for actinoallolide biosynthesis.

from Streptosporangium oxazolinicum K07-0460T (Inahashi et (Inahashi et al., 2015; Inahashi et al., 2018). The structure, al., 2011a). The structures were elucidated by NMR including the absolute stereochemistry of actinoallolide A, was spectroscopy and X-ray crystallography, and shown to be new elucidated by NMR spectroscopy and X-ray crystallography. It pyochelin family antibiotics. Spoxazomicins A-C showed was shown to be a new 12-membered macrolide that has a anti-trypanosomal activity against Trypanosoma brucei brucei 5-membered hemiacetal moiety inside the ring and a side GUTat 3.1 strain (causative agent of Nagana disease in chain including constitutive asymmetric centers (Fig. 5). The -1 animals) with IC50 values of 0.11, 0.55 and 3.37 g mL structures of actinoallolides B, C, D and E were determined by respectively. Several spoxazomicin analog have subsequently NMR spectroscopy and conversion from actinoallolide A. been reported by other groups. Li et al (2016). synthesized Actinoallolide A is potently and selectively active against analogs of spoxazomicin C and the 2-Cl-nicotinyl amide Trypanosoma brucei rhodesiense STIB900 strain (the showed activity against plant pathogenic fungi. causative agent of Rhodesiense sleeping sickness), with an -1 Tetroazolemycins A and B, dimers of spoxazomicin, were IC50 value of 0.086 g mL , as well as against T. cruzi discovered from secondary metabolites of Streptomyces Tulahuen C4C8 strain (the causative agent of Chagas disease) -1 olivaceus FXJ8.012, and showed metal ion-binding activity with an IC50 value of 0.226 g mL . and weak antibacterial activity against pathogenic Klebsiella A putative actinoallolide biosynthetic gene cluster was pneumoniae (Liu et al., 2013). Spoxazomicin D was found predicted from the draft genome sequence of strain K09-0307. from Streptomyces sp. RM-14-6 by Shaaban et al. (2017) and The gene cluster spans a contiguous 53 kb DNA region that exhibited neuroprotection activity against ethanol toxicity. comprises 7 genes encoding crotonyl-CoA reductase (aalD), 3 Spoxazomicin analogs display varied bioactivity and it seems polyketide synthases (aalA1, aalA2 and aalA3), cytochrome that phenyl oxazoline alkaloids represent a promising potential P450 (aalB), acyl-CoA dehydrogenase (aalC) and TetR family source for drug discovery. transcriptional regulator (aalR). The proposed pathway for actinoallolide biosynthesis is shown in Fig. 6. Following the 3-2. Actinoallolide generation of the polyketide backbone by AalA1, AalA2 and Actinoallomurus was recently proposed as a new genus AalA3, AalB might hydroxylate at position C-6 and (Tamura et al. 2009). Many Actinoallomurus strains have now subsequent hemiacetal formation presumably gives been isolated from plants and we have reported some new actinoallolide A. To identify the gene cluster, two cosmids species from the isolates (Matsumoto et al., 2012: Koyama et containing 27 kb (aalD, aalA1 and aalA2) and 35 kb (aalA2, al., 2012). We focused on secondary metabolites of the aalA3, aalB, aalC and aalR) regions were isolated from a Actinoallomurus strains and discovered new cosmid library of the genomic DNA of strain K09-0307. These anti-trypanosomal macrolides, actinoallolides A-E, from A. regions were assembled in a vector using Gibson Assembly fulvus MK10-036 and K09-0307, isolated from roots of and an ermE* promoter was inserted 100-bp upstream from Capsicum frutescens and Ophiopogon japonicus, respectively the start codon of aalA1. The resulting vector, pYIK3-aalM,

S60 Fig. 7. The gene cluster and proposed pathway for trehangelin biosynthesis.

was introduced into S. coelicolor M1152 and subsequent are well known as secondary metabolites of plants, the LC/MS analysis revealed actinoallolide A in the culture broth enzymes responsible for the biosynthesis have been unclear. of S. coelicolor M1152/pYIK3-aalM. Thus, the actinoallolide The in vitro enzymatic studies confirmed that ThgI catalyzes biosynthetic gene cluster was identified by heterologous the condensation of Ac-CoA and MM-CoA to expression (Inahashi et al., 2018). 2-methylacetoacetyl-CoA (MAA-CoA), ThgK catalyzes NADPH-dependent reduction of MAA-CoA to 3-3. Trehangelin 3-hydroxy-2-methylbutyryl-CoA (HMB-CoA) and ThgH Trehangelin A-C were discovered from a culture broth of catalyzes the dehydration of HMB-CoA to AN-CoA (Inahashi Polymorphospora rubra K07-0510, which was isolated from et al., 2016). This is first report on determination of the roots of Goodyera procera by physicochemical screening biosynthetic pathway to AN-CoA by using recombinant (Nakashima et al., 2013; Nakashima et al., 2017; Takahashi & enzymes. Nakashima, 2018). Trehangelin A is a trehalose angelate ester (Fig. 5) and exhibits cytoprotective properties. CONCLUSION A putative trehangelin biosynthetic gene cluster was predicted from the draft genome sequence of strain K07-0510. We have investigated endophytic actinomycetes from 29 The gene cluster spans a contiguous 16 kb DNA region that plants and more than 1,000 strains have been isolated. Most of comprises 12 genes encoding ketoacyl-[acyl-carrier-protein] them have been rare actinomycetes, which are not often (ACP) synthase (KAS III) (thgI), 3-ketoacyl-ACP reductase isolated from soil samples. Following taxonomic analyses of (thgK), enoyl-CoA hydratase (thgH), glycoside hydrolase-like the isolates, we have identified one new genus and five new protein (thgC), glycosidase (thgD), acyltransferase (thgJ), species. Furthermore, the spoxazomicins and actionoallolides transcriptional regulators (thgG and thgL), transporters (thgA which display anti-trypanosomal activities and the and thgF), membrane protein (thgE) and protein kinase (thgB). trehangelins, that exhibit cytoprotective properties, have been The genes thgHIJK were cloned in a vector pOSV556 and discovered from the culture broths of the isolated organisms. I introduced into Streptomyces albus J1074. S. albus believe that plants are an attractive and potentially as yet J1074/pOSV556-thgHIJK was incubated in YD medium untapped source for novel actinomycetes and the secondary (1.0% yeast extract and 1.0% glucose) containing 2.0% metabolites of these endophytic actinomycetes will prove to be trehalose, and subsequent LC/MS analysis revealed a useful resource for drug discovery. trehangelin A in the culture broth. This result confirms that In recent years, it has become relatively easy to obtain the thgH, thgI, thgJ and thgK are responsible for trehangelin entire genome sequence of bacteria and it has been reported biosynthesis (Inahashi et al., 2016). The proposed pathway for that 20 to 30 biosynthetic gene clusters for secondary trehangelin biosynthesis is shown in Fig. 7. ThgI, ThgK and metabolites are in the genome of one actinomycete strain (Net Thg H might be responsible for formation of angelyl-CoA et al., 2009). Utilizing the genomic information, such as (AN-CoA) from actyl-CoA (Ac-CoA) and heterologous expression of biosynthetic gene clusters and methyl-malonyl-CoA (MM-CoA). ThgC and ThgD were overexpression of regulatory genes, may help to accelerate the hypothesized to be required for the trehalose biosynthesis. discovery of more useful secondary metabolites from ThgJ might be involved in transfer of the angelyl moiety from endophytic actinomycetes and research in this respect is AN-CoA to trehalose to give trehangelin. Although angelates currently underway.

S61 antitrypanosomal antibiotics, spoxazomicins. J. Antibiot. ACKNOWLEDGMENTS 64, 297-302. Inahashi, Y., et al. (2011b). Spoxazomicins A-C, novel I would like to thank the Society of Actinomycetes Japan antitrypanosomal alkaloids produced by an endophytic (SAJ) for the Hamada Award. The results reported herein arose actinomycete, Streptosporangium oxazolinicum from research conducted in the Kitasato Institute for Life K07-0460T. J. Antibiot. 64, 303-307. Sciences, Kitasato University and I would like to express my Inahashi, Y., et al. (2012). Phytohabitans flavus sp. nov., profound gratitude to Distinguished Emeritus Professor Phytohabitans rumicis sp. nov. and Phytohabitans Satoshi Ōmura for coordinating collaboration between houttuyneae sp. nov., isolated from plant roots, and laboratories in the Ōmura Research Group. I deeply appreciate emended description of the genus Phytohabitans. Int. J. and am indebted to the supervision of and guidance of Dr. Syst. Evol. Microbiol. 62, 2717-2723. Yoko Takahashi. I would also like to express my thanks to Drs. Inahashi, Y., et al. (2015). Actinoallolides A-E, new Atsuko Matsumoto, Takuji Nakashima, Hiromi Miura, Kazuro anti-trypanosomal macrolides, produced by an endophytic Shiomi, Kazuhiko Otoguro, Masato Iwatsuki, Aki Ishiyama, actinomycete, Actinoallomurus fulvus MK10-036. Org. Toshiaki Sunazuka, Tomoyasu Hirose, Jun Oshita, Yuzuru Lett. 17, 864-867. Iwai, Haruki Yamada and all students at the Laboratory of Inahashi, Y., et al. (2016). Biosynthesis of Trehangelin in Microbial Functions. I am particularly grateful to Drs. Polymorphospora rubra K07-0510: Identification of Watanalai Panbangred, Sompop Prathanturarug, Panitch Metabolic Pathway to Angelyl-CoA. Chembiochem. 17, Boonsnongcheep and Ms. Ousana Ongcharoenwut (Mahidol 1442-1447. University, Thailand). I would also like to thank Dr. Gregory L. Inahashi, Y., et al. (2018). 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S63

Publication of Award Lecture

The Society for Actinomycetes Japan Hamada Award 2017,

Dr. Yuki Inahashi

“Isolation of endophytic actinomycetes and the search for new secondary metabolites” Actimomycetologica (2019) 33 [1], S56-S63.

Kitasato Institute for Life Sciences, Kitasato Univertsity

S64 Hamada award 2018

Biosynthesis of aminobenzoic acid derived secondary metabolites from actinobacteria

Yohei Katsuyama 1,2

1Department of Biotechnology, Graduate School of Agriculture and Life Sciences,

The University of Tokyo, Bunkyo-ku, Japan

2Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Japan

INTRODUCTION for biosynthesis of secondary metabolites, such as candicidin (Gil and Campelo-Diez 2003), trichostatin (Kudo et al. 2017) Actinobacteria produce a wide variety of natural and aureothin (He and Hertweck 2003). Anthranilic acid (o- products with useful bioactivities (Horinouchi 2007; Katz and aminobenzoic acid) is used for the biosynthesis of tryptophan, Baltz 2016). The antiparasitic agent, avermectin, is one of the but is also used as a building block of a wide variety of natural most important secondary metabolites produced by products produced by fungi and actinobacteria (Walsh et al. actinobacteria and its derivative, ivermectin, is widely used in 2012). Many aminobenzoic acids are biosynthesized from clinics. The immunosuppressant, tacrolimus, the anticancer shikimic pathway (Walsh et al. 2012). However, there are also agent, doxorubicin, and the antibiotic, streptomycin, are also alternative ways to biosynthesize aminobenzoic acid. Suzuki et clinical drugs isolated from actinobacteria. The diversity of al. reported that 3-amino-4-hydroxybenzoic acid (3,4-AHBA), bioactivities of secondary metabolites produced by which is the key intermediate of grixazone biosynthesis, is actinobacteria indicates that these organisms have acquired biosynthesized from dihydroxyacetone monophosphate and various enzymes with useful activities. Thus, understanding the aspartate-semialdehyde by two enzymes, GriH and GriI (Suzuki biosynthetic machinery is important for both basic chemistry et al. 2006). 3,4-AHBA is also the precursor of wide variety of and industrial purposes. secondary metabolites such as platensimycin, platencin Aminobenzoic acids are unusual amino acids composed (Smanski et al. 2011), nitrosobenzamide (Noguchi et al. 2010), of aromatic amine and carboxylic acid (Walsh et al. 2012). They and asukamycin (Rui et al. 2010). are important building blocks for both primary and secondary In this review, we summarize our recent discovery of novel metabolisms. For instance, p-aminobenzoic acid is used for the biosynthetic machinery used for biosynthesis of aminobenzoate biosynthesis of an important cofactor, folic acid. It is also used derived natural products.

A 2- O OPO3 CreI CreN O OH O O HOOC NH2 - O CreH NH2 CreL +N HO NH2 CreM N + HO HO CHO OH OH O O aspartate- dihydroxyacetone 3,4-AHBA 3,2,4-AHMBA cremeomycin semialdehyde monophosphate

NH2 CreE NO2 CreD OH HOOC HOOC N COOH COOH O aspartatic acid nitrosuccinic acid nitrous acid ANS pathway fumarate

B creA B C O D E H I F G J K L M N P 1 kb

Fig. 1. Biosynthesis pathway (A) and the biosynthetic gene cluster (B) of cremeomycin.

S65 A B

Arg325

Thr153

Asn77 Lys292

Leu154

Thr75 Asn294

Fig. 2. Tetrameric structure of CreD (A) and the active site of CreD (B). Each monomer has a different color. The blue sphere in (A) and stick and ball model in (B) indicate fumaric acids.

Discovery of a nitrous acid biosynthetic pathway expected that this gene is responsible for the activation of the responsible for secondary metabolism gene cluster. Substitution of the promoter of this gene to the A diazo group is a highly important functional group for constitutively active promoter (ermE* promoter) resulted in organic synthesis. It can be used for various useful reactions, production of cremeomycin in the heterologous host (Sugai et including 1,3-dipolar cycloadditions, alkylations, and carbene al. 2016). Next, the region that was not expected to be related insertions (Waldman and Balskus 2018). Interestingly, several secondary metabolisms was removed from the cosmid. The actinobacteria produce diazo containing natural products, such resultant cosmid harboring 16 genes was shown to be sufficient as kinamycin and cremeomycin (Nawrat and Moody 2011; Le for producing cremeomycin (Fig. 1B). Then, the deletion of Goff and Ouazzani 2014). Although, they have interesting each gene was performed based on this cosmid. The analysis of biological features, the biosynthetic machinery of a diazo group metabolites produced by S. albus harboring one of the cosmids remained unclear for a long time. To gain insight into the with the gene deletion, showed that at least four genes, CreN, biosynthetic machinery of a diazo group, we focused on CreL, CreE and CreD, are crucial for cremeomycin biosynthesis cremeomycin produced by Streptomyces cremeus (Fig. 1A) in addition to CreH and CreI, the homologs of GriH and GriI. (McGuire et al. 1995). This compound is a simple monocyclic CreN and CreL were expected to be responsible for methylation compound with a diazo group. According to their structure, it and hydroxylation of 3,4-AHBA to synthesize 3-amino-2- was predicted to be biosynthesized from aminobenzoate, hydroxy-4-methoxybenzoic acid (3,2,4-AHMBA). CreE and particularly 3,4-AHBA (Sugai et al. 2016). 3,4-AHBA was CreD was predicted to be responsible for diazo group formation reported to be biosynthesized by two enzymes called GriI and because deletion of these genes resulted in accumulation of GriH from dihydroxyacetone monophosphate and aspartate- 3,2,4-AHMBA (Fig. 1A). semialdehyde (Suzuki et al. 2006). Therefore, the first To further elucidate the functions of CreD and CreE, the biosynthetic gene cluster of cremeomycin was cloned using the recombinant protein of these enzymes was prepared using cosmid library constructed from the genomic DNA of S. Escherichia coli. Because CreE was predicted to be a flavin- cremeus by searching a gene encoding GriH and GriI homologs dependent monooxygenase, initially this enzyme was expected (Sugai et al. 2016). As a result, a putative cremeomycin to catalyze oxidation using 3,2,4-AHMBA as a substrate. biosynthetic gene cluster was discovered. This gene cluster However, this enzyme did not catalyze any reaction using 3,2,4- included genes encoding enzymes likely to be involved in AHMBA as a substrate. Because another nitrogen atom is cremeomycin biosynthesis, such as a methyltransferase and required for diazo group synthesis, we expected that the flavin-dependent monooxygenase. substrate of CreE is a compound providing this nitrogen atom. The cosmid was introduced into Streptomyces albus to Thus, the activity of CreE was tested against all possible amino prove that this gene cluster is responsible for the biosynthesis acids. As a result, CreE was shown to oxidize aspartic acid of cremeomycin. However, recombinant S. albus harboring this specifically (Fig. 1A). MS and NMR analysis showed that the cosmid did not produce cremeomycin. We expected that this product synthesized by CreE is nitrosuccinic acid. Thus, CreE gene cluster was not activated in S. albus. We focused on one was proved to be a unique FAD-dependent monooxygenase gene (creF) encoding a putative SARP (Streptomyces antibiotic catalyzing three step monooxygenation of the amino group of regulatory protein) family transcriptional activator and

S66 Ser286 Ser286 Ser286 (B) (B) (B) O- OH OH O Asn294 O Asn294 O Asn294 (B) (B) (B) - O H H2N O H2N O H2N O O O - O H2N O H2N O H2N N+ O Lys292 N+ O Lys292 O Lys292 O O (B) O O (B) N (B) O OH NH2 NH2 NH2

+ + H2N N Arg325 H N N Arg325 HN N Arg325 H 2 H (C) H (C) (C) + + + O HN O HN O HN Asp His Asp109 His237 Asp His 109 O- N 237 O- N 109 O- N 237 (C) H (C) (C) H (C) (C) H (C)

Fig. 3. Proposed reaction mechanism of CreD. aspartic acid to convert it to a nitro group (Fig. 1A). the same family as CreD (aspartame/fumarase superfamily). Because CreE catalyzes nitrosuccinic acid formation and Because the sequence of amino acid residues surrounding CreD was a putative lyase, the function of CreD was predicted Ser286 is also conserved in the enzymes belonging to the to be elimination of nitrous acid from nitrosuccinic acid. When aspartame/fumarase superfamily, the function of this residue is CreD was incubated with CreE and aspartic acid, the formation expected to be similar. In contrast, Arg325 is not conserved of nitrous acid along with fumaric acid was observed. Thus, among the aspartame/fumarase superfamily. This Arg325 CreD was proved to be an interesting lyase catalyzing beta- residue located near the alpha carbon of fumarase, which is why elimination of nitrous acid from nitrosuccinic acid. Because this residue was predicted to be a catalytic acid. However, the there had been no lyase catalyzing elimination of nitrous acid Arg residue is usually not preferred as a catalytic acid because + from nitrosuccinic acid, the reaction mechanism of the reaction of its high pKa value (it is too high to release H ). Therefore, to catalyzed by this enzyme was analyzed by solving the structures use Arg as a catalytic acid, there should be cationic environment of apo-CreD and CreD binding to fumaric acid with X-ray to destabilize the proton of Arg and lower the pKa value. crystallography and site-directed mutagenesis (Katsuyama et al. Interestingly, behind Arg325, His237 and Asp109 seemed to be 2018). CreD formed a tetramer consisting of four active sites forming a strong hydrogen bond. This hydrogen bond was surrounded by three of the four monomers (Fig. 2AB). This expected to stabilize the His237 as a cation. The cationic reaction was proposed to be stimulated by the acid-base environment provided by His was expected to lower the pKa catalysis. Ser286 and Arg325, acting as a catalytic base and acid, value of Arg325. Site-directed mutagenesis clearly showed that respectively (Fig. 3). In general, these residues are not preferred these three residues, Arg325, His237, and Asp109, are required as a catalytic base and acid. How Ser286 functions as a catalytic for the reaction. Taken together, the reaction mechanism of base remained unclear because the loop including this residue CreD was proposed as depicted in Fig. 3 (Katsuyama et al. is disordered in the structure of CreD. But this residue is 2018). proposed to be a catalytic base in several lyases belonging to The nitrous acid synthesized by CreD probably reacts

Fig. 4. Structures of desferrioxamine Is.

S67 Fig. 5. Other natural products reported to be biosynthesized using the ANS pathway. with the amino group of 3,2,4-AHMBA to synthesize davawensis wild type and the mutant were compared in various cremeomycin. An aromatic diazo group is usually synthesized culture conditions. However, no significant difference between by mixing aromatic amine with nitrous acid under acidic the wild type strain and the mutant was observed. Because this condition. Therefore, this reaction may happen gene cluster was expected to be sleeping under the laboratory nonenzymatically. Our gene inactivation experiments showed conditions, the combined culture method was used. Combined that there was no gene, which is crucial for formation of diazo culture is the method used to activate sleeping genes by co- groups other than creE and creD. In addition, our careful in vitro cultivating Streptomyces with Tsukamurella pulmonis. In the experiment indicated that CreE and CreD seemed not to be presence of T. pulmonis, there were several compounds required for diazo group formation. Thus, diazo group specifically produced by the wild type strain. These compounds formation in cremeomycin may happen nonenzymatically. were identified to be desferrioxamine derivatives with unusual However, recently, Waldman et al. indicated that CreM, which five membered rings at the terminal amino group (Fig. 4). is a putative AMP-dependent ligase, catalyzes diazo group Interestingly, these compounds did not have any diazo group formation (Waldman and Balskus 2018). However, further although the ANS pathway is required for its biosynthesis. The studies are required to elucidate the mechanisms of the diazo group formation might be formed during the biosynthesis diazotization reaction in cremeomycin biosynthesis. of these compounds to construct the unusual five-member ring. Interestingly, creE and creD homologs were widely In addition to our study, several groups reported that the ANS distributed among actinobacteria (Sugai et al. 2016). Many pathway is required for biosynthesis of secondary metabolites seemed to be surrounded by the genes related to secondary produced by actinobacteria, such as fosfazinomycin, kinamycin, metabolisms. Therefore, it was expected that nitrous acid triacsins and calcimycin (Huang et al. 2016; Liu et al. 2018; synthesized by this pathway seemed to be also used for Wang et al. 2018; Wu et al. 2018; Twigg et al. 2018) (Fig. 5). biosynthesis of other secondary metabolites and evolved These studies also indicated that the ANS pathway is used for a specifically for secondary metabolisms. Thus, we named this wide variety of secondary metabolites produced by pathway the ANS (aspartate/nitrosuccinate) pathway (Hagihara actinobacteria. et al. 2018). To discover other compounds synthesized by the ANS pathway, we attempted to elucidate the function of the Biosynthesis of benzastatin derivatives creE and creD homologs from other strains. As a result, we Benzastatin derivatives are secondary metabolites discovered novel desferrioxamine derivatives from composed of PABA and modified geranyl moiety which have Streptomyces davawensis and named desferrioxamine Is been isolated from several actinobacteria (Kim et al. 1996; Kim (Hagihara et al. 2018) (Fig. 4). First, the enzymatic activities of et al. 1997; Kim et al. 2001; Lee et al. 2007; Motohashi et al. the CreE and CreD homologs from S. davawensis were 2011). According to their structures, benzastatin derivatives can confirmed in vitro using recombinant enzymes. This be classified into three groups, linear, indoline and experiment clearly showed that these enzymes synthesize tetrahydroquinoline (Fig. 6). Benzastatins with indoline and nitrous acid from aspartic acid. Then, the gene encoding CreE tetrahydroquinoline scaffolds were expected to be homolog was inactivated. The metabolic profile of S. biosynthesized by cyclization of linear benzastatins via double

A B R2 C OH O NH2 H H H R2 N N H2N R1 R3 R1 O R2 R1 R3 O O

R1 R2 R3 R1 R2 R3 R1 R2

OMe Me H NH2 OMe Me NH2 Cl

H Me H NH2 OH Me NH2 OH

H CH2OH H NH2 Cl H OH Cl H Me OH OH OH H OH OH H H H

Fig. 6. Structures of benzastatins with linear (A), indoline (B) and tetrahydroquinoline scaffolds (C).

S68 bond epoxidation (Yoo et al. 1999). Although they possess completely abolished the production of cyclized benzastatins, interesting bioactivities and structural features, the biosynthesis indicating that both genes are required for cyclization. In of benzastatins remained unclear for a long time. contrast, inactivation of BezG decreased the yields of To identify the benzastatin biosynthetic gene cluster, the benzastatins with indoline and tetrahydroquinoline scaffolds genome sequence of one benzastatin producer, Streptomyces sp. and increased the yields of benzastatins with seven-membered RI-18, was analyzed (Tsutsumi et al. 2018). Streptomyces sp. rings. This result indicated that BezE is important for RI-18 was reported to produce several benzastatin derivatives, cyclization selectivity. BezB was assumed to catalyze the O- including virantmycin, JBIR-67, and 7-hydroxyl benzastatin D methylation of benzastatins with tetrahydroquinoline scaffolds (Motohashi et al. 2011). Because benzastatins consist of PABA after cyclization. and geranyl moiety, the gene cluster contains genes related to To further elucidate the biosynthetic pathway, in vitro PABA biosynthesis and prenyltransfer reaction. As a result, a analysis of recombinant BezA, BezC, BezE, BezG and BezJ single biosynthetic gene cluster was discovered as a candidate. was performed (Tsutsumi et al. 2018). In vitro analysis of BezA Heterologous expression of the gene cluster clearly showed that and BezC showed that the actual substrate of these enzymes are this cluster is responsible for the biosynthesis of benzastatins. geranyl pyrophosphate (GPP) and methylgeranyl Next, the function of each gene was analyzed by gene pyrophosphate (MGPP) (Fig. 7A). In vitro analysis of BezJ did inactivation experiments (Tsutsumi et al. 2018). Through these not provide any information because the activity of this enzyme experiments, the overall biosynthetic pathway of benzastatins could not be observed. Therefore, the function of BezJ was was deduced. During these experiments, novel benzastatin deduced by a feeding experiment using a putative product of derivatives with seven-membered rings were characterized and BezJ. Because BezJ is a homolog of AurF, which catalyzes named benzastatin K and methylbenzastatin K (Fig. 7). This in oxidation of aromatic amine, BezJ was assumed to synthesize vivo experiment showed that BezA (methyltransferase) and p-hydroxyaminobenzoic acid (PHABA). The feeding of BezC (cytochrome P450) are responsible for methylation and PHABA to the bezJ strain resulted in production of cyclized oxidation of the geranyl group before cyclization. The benzastatins, supporting the hypothesis (Fig. 7B). Next, BezG cyclization reaction was likely to be mediated by three enzymes, was shown to catalyze O-acetylation of PHABA to synthesize BezE (cytochrome P450), BezG (acetyltransferase) and BezJ N-acetoxy-p-aminobenzoic acid (PAcABA) (Fig. 7B) (AurF-like N-oxygenase). The inactivation of BezE and BezJ

A PPO BezA PPO BezC PPO

GPP MGPP HMGPP

B OH OAc C NH2 NH2 BezF R1 NH2 NH NH BezJ BezG HO HO HO HO HO O O R O O O GPP 2 PABA MGPP R1=R2=H PABA PHABA PAcABA HMGPP R1=Me, R2=H R1=Me, R2=OH

D OAc OAc H N NH BezF NH If R =H R1 R1 2 NE HO HO HO O O GPP O R2 MGPP PAcABA geranylated PAcABA R1=H: benzastain K HMGPP R1=Me: methylbenzastain K R1=R2=H If R =H R1=Me, R2=H 2 BezE OH R1=Me, R2=OH H H If R2= OH or N NE If R2= OH NE (weak) BezE HO R1 O OH OH H H R =H: JBIR-67 N N 1 NE R1=Me: 7-hydroxyl benzastain F HO HO OH Cl O O O-demethylvirantmycin Fig. 7. Biosynthetic pathway of benzastatin derivatives. BezB BezB (A) The modification of GPP by BezA and BezC. (B) The O O H H modification of PABA catalyzed by BezJ and BezG. (C) N N NE Biosynthesis of linear benzastatins by BezF. (D) HO HO Biosynthesis of cyclized benzastatin derivatives. NE: OH Cl nonenzymatic. O O 7-hydroxyl benzastatin D virantmycin

S69 OH H H 2+/3+ N Fe O 10 Fe4+/5+ 9 O R R =H HO R 2 AH 2 1 N R2 H N R1 N 10 O R1 = H HO HO 9 R1 = Me HO -OH R1 R1 OH O AcOH O H R - 2 O Cl R =OH N 2 10 HO 9 Cl O

Fig. 8. Putative reaction catalyzed by BezE.

(Tsutsumi et al. 2018). According to these results, BezE was understanding of biosynthetic pathways inspiring another field assumed to catalyze the cyclization of geranylated PAcABA of chemistry. assembled by BezF (UbiA-type prenyltransferase) (Fig. 7D). Linear benzastatins were assumed to be biosynthesized by BezF, CONCLUSION as depicted in Figure 7C. In vitro reaction using BezE clearly showed that this enzyme catalyzes the cyclization of We have discovered two novel biosynthetic machineries geranylated PAcABA. In addition, the cyclization specificity of from secondary metabolites produced from aminobenzoic acid. BezE changed according to the modification of the geranyl First, the biosynthetic pathway of cremeomycin was elucidated group. If there was no hydroxy group, BezE catalyzed the and the nitrous acid biosynthetic pathway (ANS pathway), synthesis of the indoline scaffold (Fig. 7). In contrast, if there which is responsible for diazo group biosynthesis, was was a hydroxy group, BezE catalyzed the synthesis of the discovered (Sugai et al. 2016). This is the first report that tetrahydroquinoline scaffold. In addition, when BezE catalyzed clarifies the source of the distal nitrogen atom of a diazo group the synthesis of the tetrahydroquinoline scaffold, halogenated in natural products. Interestingly, the ANS pathway seemed to benzastatin (O-demethylvirantmycin) was also observed, be used for biosynthesis of other secondary metabolites as well indicating that BezE may also catalyze nucleophilic (Hagihara et al. 2018). The distribution of the ANS pathway halogenation. indicated that nitrous acid may be used for biosynthesis of a Among the enzymes involved in benzastatin biosynthesis, wide variety of secondary metabolites and should facilitate the BezE seemed to catalyze the most interesting reaction. understanding of biosynthesis. According to the amino acid sequence, BezE belongs to a Second, a unique cytochrome P450, BezE, which cytochrome P450. Recombinant BezE possessed a heme and catalyzes the cyclization reaction via nitrene formation and showed a CO-binding spectrum characteristic for cytochrome transfer, was discovered from benzastatin biosynthesis P450s. However, BezE did not require any electron donor to (Tsutsumi et al. 2019). Although, the artificial cytochrome P450 catalyze this cyclization reaction, indicating that BezE is not catalyzes such a reaction, a cytochrome P450 catalyzing such a acting as a monooxygenase. To get further insight into the reaction had never been reported in nature. Thus, the discovery reaction mechanisms, the substrate binding spectra of BezE was of BezE clearly expands the catalytic potential of a cytochrome observed by using geranylated PABA derivatives as substrate P450 in secondary metabolisms and provided an important analogs. As a result, BezE showed a type II spectrum shift, insight into future bioengineering. Further study on its reaction which is the indicator of the amine binding to the heme iron. mechanisms, including structural elucidation, should reveal Thus, the binding of the amine to the heme was expected to be how this enzyme acquired its unique feature. important for the catalysis (Tsutsumi et al. 2018). The secondary metabolisms of actinobacteria have been The reaction mechanism of BezE was predicted as follows extensively studied in last few decades and information on (Fig. 8) (Tsutsumi et al. 2018). First, BezE catalyzes the biosynthesis of secondary metabolites is accumulating. elimination of the acetoxy group, resulting in iron nitrenoid However, our current study indicated that researching the formation. Then, the insertion of the nitrenoid to the adjacent biosynthesis of secondary metabolites from actinobacteria still double bond, resulting in aziridine ring formation, proceeds. results in the discovery of novel chemistry and there are still Finally, the aziridine ring opening by the nucleophilic addition many things we can learn from actinobacteria. Thus, studying of HO- or Cl- results in tetrahydroquinoline and indoline the biosynthesis of secondary metabolism of this organism scaffolds. BezE is the first cytochrome P450 catalyzing nitrene seems to be still important. Further study should facilitate the transfer discovered in nature. Such a cytochrome P450 had not discovery of enzymes with interesting chemistry that are been discovered in nature, but there are some cytochrome P450 important for industrial purposes. mutants that have been constructed by mutagenesis and directed evolution, which catalyzes nitrene transfer reactions (Farwell et ACKNOWLEDGEMENTS al. 2015). Most of these enzymes utilize an azide and N2 as a source and a leaving group, respectively, for iron nitrenoid These studies were carried out at the Graduate School of formation. Recently, the group of Arnold, who won the Nobel Agricultural and Life Sciences, The University of Tokyo. I am prize, recently developed enzymes catalyzing a nitrene transfer very grateful to Professors Sueharu Horinouchi and Yasuo reaction using pivalic acid as a leaving group for nitrene Ohnishi, for their support and helpful discussions. I thank all formation, which they called a nature-inspired approach (Cho my co-workers for their cooperation. I also would like to thank et al. 2019a; Cho et al. 2019b). This is a good example of an all the members in the Laboratory of Fermentation who

S70 participated in this work. This research was supported by the compounds: Biosynthesis, isolation, biological activities Japan Society for the Promotion of Science [grant number and synthesis. Bioorg. Med. Chem. 22, 6529–6544. 25850048, 25108706, 17H05432 and 18H02144], the NC- Lee, J.-G., et al. (2007). Differential antiviral activity of CARP project from the Ministry of Education, Culture, Sports, benzastatin C and its dechlorinated derivative from Science and Technology of Japan (MEXT), CREST, JST, JSPS Streptomyces nitrosporeus. Biol. Pharm. Bull. 30, 795– A3 Foresight Program and Amano Enzyme Inc. 797. Liu, X., et al. (2018). Reconstitution of kinamycin REFERENCES biosynthesis within the heterologous host Streptomyces albus J1074. J. Nat. Prod. 81, 72–77. Cho, I., et al. (2019a). Site-selective enzymatic C‒H McGuire, J. N., et al. (1995). Cremeomycin, a novel amidation for synthesis of diverse lactams. Science 364, cytotoxic antibiotic from Streptomyces cremeus. 575–578. 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S71

Publication of Award Lecture

The Society for Actinomycetes Japan Hamada Award 2018,

Dr. Yohei Katsuyama

“Biosynthesis of aminobenzoic acid derived secondary metabolites from actinobacteria”

Actimomycetologica (2019) 33 [1], S65-S71.

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Japan Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Bunkyo-ku, Japan

S72 64th Regular Colloquium

Date: Mar. 14 (Thr), 2019 Place: Institute of Microbial Chemistry (BIKAKEN)

Program:

1. “Towards the appilaction of bacterial membrane vesicles” Masanori TOYOFUKU (Faculty of Life and Environmental Sciences, MiCS, University of Tsukuba)

2. “Production of bacterial bioactive peptides based on genome mining” Shinya KODANI (Graduate School of Science and Technology, Shizuoka University)

3. “Efforts of the Ministry of Economy, Trade and Industry in the pharmaceutical field” Moriyuki HAMADA (Bio-industry Division, Ministry of Economy, Trade and Industry)

4. “Data science towards complete analysis of secondary metabolic pathways” Shigehiko KANAYA (Graduate School of Science and Technology & Data Science Center, Nara Institute of Science and Technology)

5. “Development of automated single cell analysis and isolation system: Application for microbiological research” Shun’ichi KURODA (Department of Biomolecular Science and Reaction, ISIR, Osaka University)

S73 65th Regular Colloquium

Date: May 24 (Fri), 2019 Place: Satoshi Ōmura Museum, University of Yamanashi

Program:

1. “Study of unconventional culture to access the fungal unstudied genes” Daisuke HAGIWARA (Faculty of Life and Environmental Sciences, University of Tsukuba)

2. “Molecular bases of gut symbiosis in insects” Yoshitomo KIKUCHI (Hokkaido Center, AIST)

3. “Why do majority of environmental microorganisms resist cultivation?” Yoshiteru AOI (Graduate School of Integrated Sciences for Life, Hiroshima University)

4. “Isolation and identification of Genera Kitasatospora and Putalibacter” Yōko TAKAHASHI (Kitasato Institute for Life Sciences, Kitasato University)

5. “Food development using the material made in a prefecture and tasting of wine” Fujitoshi YANAGIDA (The Institute of Enology and Viticulture, University of Yamanashi)

S74

The 2019 (34th) Annual Meeting of the Society for Actinomycetes Japan

Chairperson: Tohru Dairi (Hokkaido University, Sapporo)

The 2019 annual meeting of SAJ (SAJ34) will be held in September 2019 in Sapporo, Japan. We are looking forward to welcoming you to participate in the meeting and to submit papers. Updated information will be provided on the following SAJ34 Website: https://www.eng.hokudai.ac.jp/labo/tre/saj34/SAJ34_en.html

General Outline

Dates: September 23 (Mon)-24 (Tue), 2019 Venue: Hokkaido University Conference Hall (Sapporo Campus, https://www.global.hokudai.ac.jp/) Address: N8 & W5, Kita-ku, Sapporo, Hokkaido, Japan TEL: +81-11-706-2042

Registration fee (including abstracts): SAJ member 10,000 yen (8,000 yen until July 19, 2019) Student 5,000 yen (4,000 yen until July 19, 2019) Non-member 12,000 yen (10,000 yen until July 19, 2019) Abstracts only 2,000 yen

Registration is acceptable through the SAJ34 Website.

Banquet: From 18:30 on September 23 (Mon) 2019 at HOTEL MYSTAYS Sapporo Aspen N8 & W4-5, Kita-ku, Sapporo, Hokkaido 060-0808, Japan (Tel; +81-11-700-2111)

SAJ member 10,000 yen (8,000 yen until July 19, 2019) Student 5,000 yen (4,000 yen until July 19, 2019) Non-member 12,000 yen (10,000 yen until July 19, 2019)

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

Submission of abstracts: Abstracts for contributed oral/poster sessions should be submitted by contacting the SAJ34 organizing office.

For further information, contact to: The SAJ34 organizing office c/o, Prof. Tohru Dairi, Faculty of Engineering, Hokkaido University Address: N13 & W8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan Fax: +81-11-706-7118; E-mail: [email protected]

S75 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.

Please note; Unique set of Membership number and password is issued and provided to each eligible members of SAJ. Members are not allowed to distribute this information to the third person or third parties. Depending on the network environment there's a case where access to full contents is not permitted even though Membership number and password is correct. In such case please contact us by email for alternative access method. When contacting please provide your membership number and password, and specify name and version of your Internet browser.

RBA Helpdesk- The Journal of Antibiotics E-mail : [email protected]

S76

日本放線菌学会誌

会報

第 33 巻 1 号

— 目次 —

巻頭言「働き方改革と研究」大西康夫･･････････････････････････････････････ 2 2018 年に正式発表された放線菌学名一覧について（解説）･････････････････････････ 3

受賞論文のお知らせ･･････････････････････････････････････････････････････････ 4

小山泰正先生を偲んで･･･････････････････････････････････････････････････････ 5 2018 年度日本放線菌学会企業賞受賞論文

（アステラスファーマテック株式会社富山技術センター）･･････････････････････ 8

2019 年度（第 34 回）日本放線菌学会大会のご案内･･･････････････････････････････ 16 2019 年度日本放線菌学会四賞授賞者の決定について･････････････････････････････ 18

第 64 回日本放線菌学会学術講演会･････････････････････････････････････････････ 19 第 65 回日本放線菌学会学術講演会･････････････････････････････････････････････ 28

日本放線菌学会賛助会員･･････････････････････････････････････････････････････ 37 著作権について･･･････････････････････････････････････････････････････････････ 37

1 巻頭言

働き方改革と研究

平成 31 年 4 月より関連法案の一部が施行され、令和の時代の到来とともに「働き方改革」が本格的に進もうとしている。人口減少、超高齢化社会の到来が確実な我が国において、労働力人口確保のためには多様な働き方を可能にしつつ労働生産性を上げることが不可欠だというのが、その根底にあるようだ。働き方改革の三本柱のうち、「非正規と正社員の格差是正」、「高齢者の就労促進」については大きな異論はないが、「長時間労働の改善」に関しては、研究・教育に携わる者として、やや心配に思うところがある。常日頃から学生には思う存分実験に打ち込んでもらいたいと思っているのだが、世の中全体が「働きすぎは悪」のようなムードになると、そもそも時間がかかる生命科学実験系の研究に取り組もうと思う若者が減ってしまうかもしれない。また、その道を選んだとしても、定時に帰れないような実験はしたくないという人が増えるかもしれない。例えば、近い将来、3 日間 6 時間おきにサンプリングするといったタイムコース実験を一人に任せることはできない時代になっていくのかもしれない。時間さえかければいい研究ができるということは決してないが、ある一定以上の時間をコンスタントに実験に費やすことは、いい研究を生み出す要素の一つではないだろうか。解明したい「謎」にいろいろな角度からアプローチすることは、その研究が独創的であればあるほど重要となり、求められる実験量は増えていくものだ。新しい実験にはさまざまな試行錯誤も必要である。素晴らしい研究成果は膨大な実験量によって支えられていることがほとんどであるように思う。学生ではなく研究を職業としている者はどうだろうか。いわゆるワークライフバランスは重要であるが、働き方は多様であるべきで、とことん研究に打ち込む者がいてもよいだろう。本人の意思に反して、制度のために研究が時間的に制限されるというのは「科学技術立国」にはふさわしくないように思う。そうは言っても、特に企業ではいろいろ難しいところもあるだろう。効率化を重視したスタイルは当然であろうが、それで研究の本質が変わることがあってはならないと思う。世の中の誰もが知らないことを初めて明らかにする楽しさがあってこそ研究であり、それがなければ実験はただの作業となる。本学会員の研究対象である放線菌一つをとってみても、まだまだわからないことは山のようにある。そのような「謎」を自らの手で 1 つ１つ解明していく仕事は本当に魅力的である。「働き方改革」が進む中、今後、研究者はこの研究の本質的な魅力をこれまで以上の熱意をもって若手・学生に伝えていく必要があるように思う。

2019 年 6 月 17 日日本放線菌学会副会長大西康夫（東京大学大学院農学生命科学研究科）

2 2018 年に正式発表された放線菌学名一覧について

― List of new scientific names and nomenclatural changes in the phylum Actinobacteria validly published in 2018 ―

日本放線菌学会の菌株基準委員会では、毎年 Actinomycetologica 第 1 号で、前年中に正式発表された放線菌（phylum Actinobacteria）の学名をその基準属（type genus）/基準種（type species）/基準株（type strain）や文献（reference）とともに一覧にして紹介しています。昨年は、新たに 2 目（orders）、8 科（families）、29 属（gerera）、151 種（species）、20 亜種（subspecies）が承認（Int J Syst Evol Microbiol 誌に学名が掲載）されました。ゲノム配列を用いた Nouioui らの広範囲に渡る放線菌の再分類（Front Microbiol., 2018, 9: 2007）により、亜種から種への格上げ（rank elevation）による新種（new species）も散見され、新亜種（new subspecies）の大半は種から亜種への格下げ（rank reduction）によるものでした。また、再分類により 200 以上の種が他の属に移行しました。それに伴う学名変更が new combination や new nomenclature に含まれています。これら一覧ではバソニム（basonym）として移行前の学名が載っています。例えば S44 ページ new combination の最後に示した「Yinghuangia aomiensis (Nagai et al. 2011) Nouioui et al. 2018, comb. nov.」ではバソニムが Streptomyces aomiensis Nagai et al. 2011 なので、Streptomyces aomiensis が Streptomyces 属から Yinghuangia 属に移行し、学名が Yinghuangia aomiensis になったことを意味します。Nouioui らの研究や同じくゲノム配列を用いた Gupta らによる Mycobacterium 属の再分類（Front Microbiol., 2018, 9: 67）などにより、昨年よりも new combination の数が大幅に増えました（下表参照）。 Emendation とは、その分類群を定義している性質の記載（description）が追記・修正されたことを意味します。具体的な追記・修正内容は各 reference をご確認ください。別種として異なる学名で承認されていた複数種が同種の場合、それらをシノニム（synonym）と呼びます。 Mycobacterium africanum 、 Mycobacterium bovis 、 Mycobacterium caprae 、 Mycobacterium microti、Mycobacterium pinnipedii、Mycobacterium tuberculosis の 6 種がシノニムとなり、これらの中で最も古い学名である M. tuberculosis が優先されます。Streptomyces ciscaucasicus と Streptomyces canus もシノニムとなり、学名としては S. canus が優先されます。本誌 SAJ News の S3 ページからは、以下の表の項目順に 2018 年に正式発表された放線菌の学名一覧を掲載しています。

正式発表数 2017 年 2018 年 New order 0 2 New family 6 8 New genus 15 29 New species 168 151 New subspecies 1 20 New combination 16 212 New nomenclature 0 5 Emendation of family 2 1 Emendation of genus 8 6 Emendation of species 17 24 Synonym 7 6

菌株基準委員会・委員長小牧久幸（製品評価技術基盤機構）

3 受賞論文掲載のお知らせ

2018 年度日本放線菌学会企業賞受賞アステラスファーマテック株式会社富山技術センター（竹下敏一氏）

「放線菌 Streptomyces tsukubensis が生産するタクロリムスの安定供給および増産への取り組み」

"Efforts of stable supply and increasing production for tacrolimus produced by Streptomyces tsukubensis"

Toyama Technology Center, Astellas Pharma Tech Co., Ltd.

Mr. Toshikazu Takeshita

日本放線菌学会誌（2019）33[1], 8-14

4 小山泰正先生を偲んで

日本放線菌学会名誉会員であられます東邦大学名誉教授小山泰正先生が、平成 30 年 9 月 10 日、享年 87 歳にてご逝去されました。先生は、京都府にお生まれになり、京都大学医学部薬学科に入学、同大学大学院修士課程（生薬学）を修了された後、千葉大学腐敗研究所（抗生物質研究部）の助手として赴任、昭和 39 年 4 月には同大学薬学部微生物薬品化学教室の専任講師、昭和 44 年 9 月に助教授に昇任されました。先生の放線菌研究は千葉大学腐敗研究所の新井正教授の下、抗生物質の単離を中心に始まりました。また、腐敗研究所に導入されたばかりの赤外分光光度計を用いた「全菌体 IR スペクトルによる放線菌の分類」というユニークな研究も行われました。放線菌の二次代謝産物を中心にした研究業績の一部を「Streptomyces luteoreticuli の代謝産物に関する研究」としてまとめられ、京都大学大学院薬学研究科より薬学博士の学位を取得されました。そして、昭和 51 年 4 月から東邦大学薬学部微生物学教室の教授として赴任され、ご定年により退職された平成 8 年 3 月までの 20 年間、放線菌を中心とした微生物の多彩な機能を導き出す研究、例えば、光誘導カロチノイド合成、抗変異原性物質の探索、 Nocardia 属ならびに Micromonospora 属の宿主ベクター系の確立、Micromonospora griseorubida が生産するマクロライド系抗生物質マイシナミシンの生合成などを行われました。また、先生は私立大学薬学部にとって最も重要な薬剤師育成教育にご尽力なされる傍ら、

5 学校法人東邦大学の評議員や理事、東邦大学習志野図書館長、更に昭和 60 年 4 月から昭和 63 年 3 月には東邦大学薬学部長を務められるなど、大学ならびに法人の運営にも携われ、先生の類まれなる企画力、行動力、指導力、そして何よりもその人望により、大学ならびに学部運営に様々な改革をなされました。先生は、本学会の設立やその後の運営に多大なる貢献をなされました。昭和 60 年に本学会の前身である放線菌研究会が日本学術会議の学術登録団体となり、岡見吉郎先生を初代会長として発足した日本放線菌学会は、平成 2 年に放線菌育種談話会（当時：代表幹事岡西昌則先生）と合流して現在の本学会となりましたが、先生は、合流の際には本学会の学会長としてご尽力され、新生日本放線菌学会の学会長を別府輝彦先生にバトンタッチされました。昭和 63 年の ISBA’88（第 7 回国際放線菌学会議）では組織委員や Proceedings “Biology of Actinomycetes ’88” (Okami Y, Beppu T, Ogawara H, 1988)、Trends in Actinomycetology in Japan –Actinomycetologica Forum– (Koyama Y, 1989)の刊行に貢献され、翌年 1 月の日本学術会議微研連シンポジウム「ACTINOMYCETES 昨日・今日・明日」を主宰されました。その後、平成 7 年には学会設立 10 周年記念大会となる日本放線菌学会大会（東京）の大会長として大会の運営にあたるともに、国際シンポジウム「Emerging Research Targets in Actinomycete Secondary Metabolite」の開催にご尽力されました。このような本学会への多大なる貢献から、平成 3 年には本学会功績功労賞「日本放線菌学会交流基盤の充実・整備」を受賞され、平成 17 年には本学会名誉会員となられました。私は昭和 62 年の 4 月から卒研生として先生の研究室に所属させて頂いたのですが、当時、先生の研究室はとても人気があり、薬学部の 20 名ほどの 4 年生が卒研生として配属されていたにも関わらず、私を含めて理学部の 3 名もの学生を受け入れてくださいました。この年は先生が本学会の会長に就任された年であり、また、薬学部長でもあったため、多忙極まりない中、常に学生にも目を向けてくださりました。私は、更に修士課程の 2 年間を含めた 3 年間を学生としてお世話になり、その間、幸運にも ISBA’88 や日本学術会議微研連シンポジウムを経験させて頂いたのですが、その場面ごとに学生が活躍する場を与えて、常に学生の特性を見極め、研究ばかりでなく、様々な面での学生への教育をされていました。この様に、先生は常に学生のことを第一にお考えになる「学生ファースト」を実践されていたため、多くの学生、更に大学関係者から慕われておりました。それは卒業生に対しても同じであり、私は、修士課程修了後、製薬会社にて放線菌を中心に研究を行っていたこともありますが、学会等でお会いするたびに激励のお言葉を頂き、それは 20 年前に研究室に戻ってきてから、更に、微生物学教室前教授の加藤文男東邦大学名誉教授から研究室を引き継がせて頂いてからもお気遣い頂いておりました。先生は、単に、優しく、気配りをされていたのではなく、時に厳しく、また、揺るぎない信念をもって卒業生に対しても接していらっしゃいました。そのためか、当研究室では研究室の同窓会である「伍の会」を 5 年ごとに開催していますが、毎回、参加者が 200 名を超えております。先生がお亡くなりになられたのは、奇しくも平成 30 年度の本学会大会の前日であった

6 ため、早々に早川正幸学会長をはじめ数名の先生方に訃報をお伝えすることが出来ました。これは全くの偶然ではありますが、これも先生のお気遣いだったのかも知れません。そして、半年後の平成 31 年 3 月 16 日に「小山泰正先生を偲ぶ会」を開催させて頂き、本学会の諸先生方にも多数ご参列頂きましたこと、この場をお借りして心より御礼申し上げます。先生の厳しいご指導の中で、時折見られる笑顔を見ることが出来ないのは寂しいです。先生のご教導に心より感謝申し上げます。謹んでご冥福をお祈り申し上げます。

（東邦大学薬学部微生物学教室教授安齊洋次郎）

7 2018 年度日本放線菌学会・企業賞受賞総説

放線菌 Streptomyces tsukubensis が生産するタクロリムスの安定供給および増産への取り組み

竹下敏一

アステラスファーマテック株式会社富山技術センター〒930-0809 富山県富山市興人町 2 番 178 号

Efforts of stable supply and increasing production for tacrolimus produced by Streptomyces tsukubensis

Toshikazu Takeshita

Toyama Technology Center, Astellas Pharma Tech Co., Ltd. 2-178 Kojinmachi, Toyama-shi, Toyama 930-0809

1．はじめに雑な代謝ネットワークの理解が十分とはいタクロリムスは、アステラス製薬（当時のえないまま商用製造を迎えることとなる。藤沢薬品）において新規に分離した放線菌しかし、商用製造では増産や培地原料の品 Streptomyces tsukubensis No.9993 により生産質変動・終売対応がある。生物を利用した医される二次代謝産物である（図 1）。タクロ薬品生産プロセスでは増産や原料の品質変リムスを有効成分とするプログラフ製品は、動・終売対応は製品へ直接影響を及ぼさず、優れた免疫抑制機能から臓器移植における生物の代謝に影響を及ぼす。その結果とし拒絶反応の抑制などに使われる免疫抑制剤て目的物質、構造類縁体に影響が現れる。故として約 100 の国と地域で販売され、世界に生産菌の代謝ネットワークを如何にしての移植領域の分野で広く貢献している。理解し、制御するのか、その戦略が重要となタクロリムスは一次代謝産物であるシキる。ミ酸、アセチル CoA、ピペコリン酸を原料当社富山技術センターではプログラフとして、生産菌の二次代謝により生合成さ製品の発売以降、25 年以上にわたって安定れる。一般的には、医薬品製造の工業化研究的にタクロリムスを製造し続けてきた。本に要する時間は限られており、生産菌の複稿ではその過程で培ってきた微生物発酵の

8 制御戦略について扱う。事例として「構造類 2．計測データ活用による二次代謝メカニ縁体 FR900525 の生成メカニズムの推定とズムの理解生産菌育種への展開 1」、「培地原料の品質変タクロリムス製造法の開発当初は、まだ動が招いた低生産の改善」の 2 例を挙げる。オミクス解析の黎明期であった。そこで、筆者らは生産菌の二次代謝メカニズムを解明するにあたって、発酵工程の計測機器から得られる様々な数値と制御したい目的化合物や性質との関係性を丹念に探ることで、生産菌の代謝を推定し、制御する最適な方法を探索してきた（図 2）。計測データの例としては、①発酵槽の制御に利用されるオンラインデータ（pH、DO（溶存酸素濃度）、温度等）、②発酵槽からサンプルを採取して測定するオフラインデータ（PMV（Packed Mycelium Volume）、粘度等）③発酵液上清を HPLC で分析して得られる代謝物質濃度、が挙げられる。制御の対象は、タクロリムス図 1. タクロリムス（FK506）の構造式や構造類縁体などの化合物の生産量や、生産性に影響を与える発酵液の液性や呼吸活性などである。これら計測データを微生物の代謝を表す指標として考え、制御対象と

Packed MyceliumMycelium VolumeVolume

INPUT Step 2 Step 1 OUTPUT 培地・培養条件発酵計測データ制御対象

操作菌糸形態内圧 DO 糖 pH タクロリムス培地原料アミノ酸 OUR 温度 EX-CO2 構造類縁体有機酸 pH 流動

図 2. 計測データを利用した二次代謝メカニズム理解のためのストラテジー Step 1 制御対象と計測データの関係性を探り、重要な計測項目を特定する Step 2 重要計測項目を指標として培地・培養条件を調整し、制御対象の制御を実現する

9 の関係性を導き出し、重要な計測項目を特性により精製工程での除去が困難であった定する。そして、重要な計測項目を指標としため、発酵による生産過程で低減する必要て培地・培養条件を調整し、制御対象を意図があった。する方向へ制御するのである。微生物発酵は変動性が大きい。実験室規模であっても実験ロット間でデータの再現性が乏しく、化合物生産量の制御が困難な場合がある。また、工業化研究により開発した製造法であっても商用製造を重ねるにつれて、開発段階では経験のない変動に直面することがある。こうした変動は全く同じ条件で実施されているにも関わらず起きるため、変動性の原因究明は一見困難と思える。しかし、発酵工程から得られる計測データを丁寧に解析することで、変動性の原因、そして制御すべき真のパラメータを探索することができることを筆者らは経験してきた。限られたデータ数だけでは変動の実態をとらえることは困難である。しかし、実験室、商用製造規模から得られるデータを回顧的に解析することで変動性を超えた真実に近い方向性が浮かび上がってくる。こうした取り組みを繰り返しながら、タクロリムス発酵のメカニズムを理解・制御し、増産図 3. FR900525 の構造式と安定供給を実現してきた。上段：タクロリムス、下段：FR900525

3．生産菌育種による FR900525 生成抑制まず、実際の発酵において、FR900525 の菌株育種は目的物質の生産性を向上させ生成量を制御している要因を調査した。る手段の 1 つである。しかし、菌株育種で FR900525 生成量の異なる発酵液サンプル得られる生産性向上株は目的物質だけでなを用いて、上述の計測データ、特に上清中のく構造類縁体を増加させる負の側面もある。代謝物質としてアミノ酸、有機酸から、その 1 例が FR900525 である（図 3）。 FR900525 と相関する成分のスクリーニン FR900525 はタクロリムスの分子骨格のピグを実施した。その結果、メチオニンを除くペコリン酸部位がプロリンに置換された構アスパラギン酸ファミリーのアミノ酸群と造類縁体である。FR900525 はその構造類似 FR900525 は負の相関があり、特にタクロリ

10 ムス原料であるピペコリン酸が強い負の相関を示した。一方で、当初の予想とは異なり FR900525 はプロリン濃度と正の相関を示さなかった。以上の結果から、生産性向上株ではタクロリムス生合成系が増強され、ピペコリン酸取り込み量が増加するが、ピペコリン酸の生合成が追い付かず、誤ってプロリンが取り込まれることで、FR900525 が図 4. AEC 濃度勾配プレート（左：低濃度、右生成される、というメカニズムが推定され高濃度）で取得した耐性株と親株の生育を比較た。（上：親株、下：耐性株）その仮説から、生産菌のピペコリン酸生親株は AEC 濃度依存的に生育阻害を受けるが合成能を増強する手段として、リジンアナ耐性株は濃度によらず生育が認められたログである AEC の耐性株を得ることで、ピペコリン酸の前駆体であるリジンの生合成量を高める方法を採用した。得られた耐性表 1. 親株と AEC 耐性株の発酵結果株（図 5）は親株と比較して、培養上清中のピペコリン酸濃度が増加し、FR900525 の低減が認められた（表 1）。結果的にタクロリ

ムス生産量も増加していた。これは原料で FR900525 生成の予兆である。ピペコリン酸あるピペコリン酸生合成量の増加が寄与しの挙動から検知することで早期に対策を講たと考えられた。じることができる。この関係性は商用製造での FR900525 生以上が培養上清中の成分と構造類縁体の成のリスクモニタリングにも活用できる。関係性を明らかにし、生産菌の育種へ応用もし何らかの原因でピペコリン酸濃度が低した事例である。筆者らは培養上清中の代下する製造ロットが頻発すれば、それらは謝物質は菌体内より容易に測定できるため、

図 5. ピペコリン酸（縦軸）と FR900525 生成量（横軸）の関係ピペコリン酸濃度が 0.3 mM を下回ると急速に FR900525 が生成される

11 間接的ではあるが生産菌の代謝を理解する成分に注目しただけでは、原料品質の変動一助となると考えている。を捉えることはできない。そこで、複数成分による多次元的な評価をするために多変量 4. 培地原料の品質変動が招いた低生産解析を採用した。解析の対象は 3 つの窒素の改善源とし、原料の品質を表す項目にはメーカタクロリムスの発酵培地には炭素源、窒ー提供の試験結果および自社試験結果を利素源として多くの天然物由来の原料が使わ用した。解析に用いた原料のロット数は窒れている。天然物由来の原料を使用するメ素源 A が 64 ロット、窒素源 B が 22 ロッリットは、①安価であること、②アミノ酸、ト、窒素源 C が 40 ロットである。ビタミン等各種成分が豊富に含まれること、重回帰分析により、これらのデータを用 ③不溶性粉末が微生物の生育に応じて緩やいて発酵生産量を予測するモデルを構築しかに消費されるため流加培養無しで高密度たところ窒素源 A のモデルから予測された培養が可能となること、が挙げられる。しか生産量と実際の生産量が相関することが示し、出発原料に作物を使用しているため、産された（図 7）。構築されたモデルからは生地や天候により原料の品質が変動しやすく、産量に寄与する窒素源 A の重要項目が推定原料の製造ロットにより発酵生産量、構造され、これら重要項目の変動が商用製造で類縁体量が変動してしまうことが経験的にの生産量変動・低下の原因と考えられた。わかっている。筆者らは、長期にわたるタクロリムスの商用製造において培地原料の品 4-2 製造データのトレンド解析質変動による発酵生産量の変動および低下発酵槽には各種センサが取り付けられてを確認してきた。そのため質の悪い発酵培おり、すべての製造において連続的に計測地原料を用いても、需要に応えられるよう、データが記録され、トレンドデータとしてプロセスの改善を試みた。以下にその取り収集されている。商用製造でも pH、DO、排組みを紹介する。出ガス濃度など多種類のトレンドデータを蓄積している。トレンドには大なり小なり 4-1 原料品質の解析変動がある。トレンドの変動と制御対象と天然物由来の原料は成分が複雑で特定のの相関関係を調査することがトレンド解析

図 6. 重回帰分析法による培地原料の品質解析結果予測値と実際の生産量との関係

12 である。態によって発酵生産量が変化することがわ商用製造 83 ロットのトレンドデータとかった。そして、シミュレーションにより最発酵生産量との関係性を解析し、高生産発適な流動状態が得られる撹拌翼、スパージ酵の特徴を調べた（図 8）。DO と pH のトレャーを設計し、商用製造の発酵槽に反映しンドデータの変動を比較すると DO の変動た。が大きい。しかし、相関係数をみると、DO こうした取り組みの結果、培地原料の品の変動と生産量には相関関係がない。一方質変動により低下していた発酵生産量をで pH トレンドの変動は比較的小さいもの 30%向上させることができ、原料の品質変の、発酵生産量とマイナスに相関している動に左右されない堅牢な発酵プロセスとすフェイズがあることがわかった。ることができた。プロセス改善以降、原料の検証実験によりこの pH トレンドの変動品質変動による発酵生産量の変動は認めらには先に述べた原料品質の変動が影響してれていない。いることも明らかとなった。そこで、原料品質の影響に左右されず、高生産発酵特有の 5. まとめ pH トレンドを安定的に再現可能な培地・培生物を用いた医薬品生産プロセスは生物養条件を構築した。の複雑さ故に、その全容を理解し、制御することは難しいものである。そのため、筆者ら 4-3 コンピュータシミュレーションによは発酵プロセスから得られる計測値を利用る発酵槽の最適化することで生物の代謝を推測し、改善のヒ生産量改善への取り組みには上述の原ントをみつけてきた。生産物、構造類縁体と料・製造データの解析に加え、コンピュータ相関する計測項目を見出すことはプロセスシミュレーションによる流動解析を行った。最適化の方向性を正しく導くものであると商用製造の発酵槽の流動状態の定量化を行考えている。また最新技術を取り入れるこい、小スケールの流動状態と比較し、流動状とで新しい知見を得ることができた。多変態と発酵生産量の関係を調べると、流動状量解析、コンピュータシミュレーション、こ

DO pH

1.0 1.0

0.5 0.5

0.0 0.0 相関係数 -0.5 相関係数 -0.5

-1.0 -1.0 培養フェイズ培養フェイズ

図 7. 商用製造の DO、pH トレンド（上段）と発酵生産量との相関係数（下段）

13 うした技術を取り入れることは微生物発酵謝辞を高度に制御するための有用なツールにな長きにわたる生産・品質の改良・改善をり得ると考えている。成しえたのは、当社の技術開発部門、製造部門、品質管理部門に関わる各部門のメン 6. おわりにバー、そしてアステラス製薬株式会社バイ生物のもつ変動性は研究者・技術者の頭オ技術研究所のメンバーの強いチームワーを悩ませる。同じことをやっても、同じ結クによって成された成果です。携わったす果が得られないのである。こうした変動性べての方に感謝申し上げます。は商用製造のように確立された製造法であ特に、多変量解析および製造データの解ってもしばしば観測される。本稿で述べた析に関して多くの貴重なアドバイスを頂き原料の品質変動はその一部で、筆者らは増ました、加藤竜司准教授（名古屋大学）に産時や、原料メーカーの変更時に生産量のは深く感謝申し上げます。低下・構造類縁体の増加といった変動性を何度も経験してきた。しかし、その都度、発酵プロセスから得られるデータを丁寧に引用文献 1. Shimizu, S., Futase, A., Yokoyama, T., Ueda, S., 解析し、発酵の制御を確実にする術を見出 Honda, H. (2017). Reduction of FR900525 using し、製造法を堅牢なものにしてきた。生物 an S-(2-aminoethyl) l-cysteine-resistant mutant. Journal of Bioscience and Bioengineering, 123(6), の変動は複雑ではあるが、必ず原因があ 685-691. り、その原因をとことん追求する過程で生

産菌の画像解析、多変量解析、コンピュータシミュレーションといった新しい技術を獲得し、発酵生産技術を発展させてきた。こうした取り組みは微生物発酵にとどまることはなく、動物細胞、ヒト細胞の培養技術にも適用できると考えている。オミクス解析といった高度な技術だけに頼るのでなく、まずは容易に利用できる日々の計測データに丁寧に向きあうことが、生物の複雑さを理解する第一歩である。本稿がその一助になれば幸いである。

14 2018 年度企業賞受賞

アステラスファーマテック株式会社富山技術センター

「放線菌 Streptomyces tsukubensis が生産するタクロリムスの安定供給および増産への取り組み」

Toyama Technology Center, Astellas Pharma Tech Co., Ltd.

Efforts of stable supply and increasing production for tacrolimus produced by Streptomyces tsukubensis

2019 年度（第 34 回）日本放線菌学会大会のご案内

大会長大利徹（北海道大学大学院工学研究院）

2019 年度大会は、北海道大学札幌キャンパスにて 9 月末に開催することになりました。8 月から 9 月にかけては多くの学会が札幌で開催され、会場確保が年々難しくなっています。今回も一年半以上前から会場確保を試み、何とか札幌キャンパス内で確保できましたが、9 月後半の 3 連休の最終日から始まる日程となってしまいました。この時期は札幌大通公園で札幌オータムフェスタが開催され、北海道名産食材を堪能できますことから、早めに来札され楽しんでいただければと思います。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 2019 年度大会ウェブサイト(https://www.eng.hokudai.ac.jp/labo/tre/saj34/) を通じてご案内いたします。

概要

・期日: 2019 年 9 月 23 日（月）- 24 日（火）・会場: 北海道大学札幌キャンパス、学術交流会館 TEL: 011-706-2042 (https://www.hokudai.ac.jp/bureau/property/s01/) ・交通: JR 札幌駅、地下鉄札幌駅から徒歩 8 分・参加費（講演要旨集代を含む） 7 月 19 日まで 7 月 20 日～当日正会員： 8,000 円 10,000 円学生： 4,000 円 5,000 円非会員： 10,000 円 12,000 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。

講演申込、講演要旨提出、大会参加の事前申込締切日: 2019 年 7 月 19 日（金）・懇親会: 日時: 2019 年 9 月 23 日（月）18:30～20:30 (予定) 会場: ホテルマイステイズ札幌アスペン (https://www.mystays.com/hotel-mystays-sapporo-aspen-hokkaido/) 会費 7 月 19 日まで 7 月 20 日～当日正会員： 8,000 円 10,000 円学生会員： 4,000 円 5,000 円非会員： 10,000 円 12,000 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 [ショートプレゼンテーションあり]） 2. 受賞講演（大村賞・浜田賞） 3. 招待講演｛及川英秋博士（北海道大学大学院理学研究院）・阿部郁朗博士（東京大学大学院薬学研究科）｝

16 参加および講演申し込み要領

● 参加および講演申し込み（大会登録システムをご利用ください）大会ウェブサイト(https://www.eng.hokudai.ac.jp/labo/tre/saj34/)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：2019 年 7 月 19 日（金）＊大会参加の事前申し込みの締切日：2019 年 7 月 19 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。

郵便局から口座記号番号： 02700-5-103231 口座名称：日本放線菌学会第 34 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ヨンカイタイカ

他銀行から銀行名：ゆうちょ銀行店名：二七九（ニナナキユウ）店番：２７９預金種目：当座口座番号：0103231

● 発表者は学会会員に限らせていただきます。入会手続きについては学会ホームページをご覧下さい。口頭発表の希望者が多い場合、ポスター発表への変更をお願いすることがあります。ポスター発表者にはショートプレゼンテーションを行っていただく予定です。 ● 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。和文・英文どちらで作成して頂いても結構ですが、発表言語との統一（和文→日本語、英文→英語）をお願い申し上げます。所属は和文・英文とも省略形で記入してください。英文タイトル等は英文プログラム作成に使用します。2 ページ目に記載してください（英文要旨の場合には不要）。 ● 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いたします。

お問合せ先第 34 回放線菌学会大会事務局 E-mail: [email protected]

17 2019年度日本放線菌学会四賞授賞者の決定について

2019年4月12日会長早川正幸

日本放線菌学会は、下記のように2019年度日本放線菌学会四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）および日本放線菌学会企業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）】五十嵐雅之氏（公益財団法人微生物化学研究会微生物化学研究所）「多剤耐性菌に有効な放線菌の代謝物に関する探索研究」

【功績功労賞】該当者なし

【浜田賞（研究奨励賞）】以下の2名（五十音順）淡川孝義氏（東京大学大学院薬学系研究科）｢放線菌アルカロイド生合成酵素の発掘および物質生産｣

手塚武揚氏（東京大学大学院農学生命科学研究科）「胞子嚢を形成する希少放線菌の形態分化に関する分子遺伝学的研究」

【企業賞】該当者なし

以上

18 報告第 64 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 31 年 3 月 14 日（木） 13:00〜17:30

場所：微生物化学研究所別館 2 階会議室参加者： 50 名

プログラム

1．『バクテリアが放出する膜小胞の利用に向けて』豊福雅典（筑波大学生命環境系微生物サステイナビリティ研究センター）

2．『ゲノムマイニングを基盤としたバクテリアの生理活性ペプチドの生産』小谷真也 (静岡大学農学部応用微生物学研究室）

3．『医薬品分野における経済産業省の取り組み』浜田盛之 (経済産業省）

4．『二次代謝物の生合成経路の悉皆的解析を目指したデータサイエンス』金谷重彦 (奈良先端科学技術大学院大学情報科学研究科）

5．『全自動１細胞解析単離装置の開発および微生物研究への応用』黒田俊一 (大阪大学産業科学研究所生体分子反応科学研究分野)

19 バクテリアが放出する膜小胞の利用に向けて豊福雅典（筑波大学生命環境系微生物サステイナビリティ研究センター） [email protected]

多くの細菌は細胞外に数百ナノメートル程度のメンブレンベシクル (MV) を放出することが知られている。MV は微生物間コミュニケーションや遺伝子の水平伝播など、細菌の生存や進化における重要なプロセスに関与しており、宿主への病原性への発揮にも関わっていることが報告されている。また、抗生物質耐性に関わるなど、多岐に渡る機能を示し、二次代謝産物の放出にも関わることが近年分かってきた。MV の機能解明が進む一方で、MV の形成機構については、未解明な部分が多かった。従来では、グラム陰性菌においては、外膜がたわむ形で MV が形成されるとされていた (blebbing)。その一方で、我々は緑膿菌を用いた研究で、ライブセルイメージングを駆使することで blebbing とは全く異なる機構で MV が形成されることを新たに見出した。当該 MV 形成機構においては、細胞壁分解酵素 (エンドリシン)の働きによって、細胞壁を失った細胞が破裂し、その膜断片が再会合するかたちで、MV が形成されることを明らかにし、explosive cell lysis と名付けた。 explosive cell lysis を誘発する鍵となる酵素はエンドリシンである。エンドリシンは dsDNA ファージが宿主を溶菌して外に出る際に一般的に用いている酵素であり、細菌間で最も広く保存されている遺伝子の一つである。我々は、グラム陽性菌として知られる枯草菌の MV 形成にもエンドリシンが関わることを見出した。エンドリシンが関わるものの、その詳細な MV 形成過程が explosive cell lysis と異なるため、これを bubbling cell death と名付けた。さらに、エンドリシンを介した MV 形成機構はミコール酸含有細菌でも観察された。これらのことより、エンドリシンは異なる細胞構造を持つ細菌において MV 形成を誘導することが明らかとなった。MV 形成機構が明らかになったことで、MV 形成を誘導させることも可能となった。これは細菌の二次代謝産物を細胞外へ積極的に放出させる技術の構築にも繋がってくる。こうした知見は物質生産にも応用できると考えられる。

参考文献 1) Toyofuku M., et al. Nat. Rev. Microbiol. 17, 13-24, 2019. 2) Toyofuku M., et al. Nat. Commun. 8, 481, 2017 3) Turnbull L., Toyofuku M., et al. Nat. Commun. 7, 11220, 2016

20 ゲノムマイニングを基盤としたバクテリアの生理活性ペプチドの生産小谷真也（静岡大学学術院農学領域） [email protected]

カビや細菌等の微生物は多種多様な生理活性ペプチドを生産することで知られている。それらのペプチドは生産システムによって、大きく分けて 2 種類になる。非リボソームペプチド（non- ribosomal peptide）もしくは、リボソーム翻訳系翻訳後修飾ペプチド(Ribosomally synthesized and post-translatinally modified peptides)である。近年の次世代シーケンサーによる遺伝子配列の決定技術により、データベースの遺伝子情報が非常に速いスピードで蓄積されつつある。ゲノムマイニングとは、ゲノム情報から生合成に関する情報を検索し、それを実際の有用物質の生産に結び付ける技術である。膨大な遺伝子情報からゲノムマイニングで新規ペプチドを探索する試みが世界中で展開されつつある。発表者は、近年精力的にこの技術を用いたペプチド探索および異宿主生産の研究に取り組んでおり、その成果を発表する。

1．ゲノム情報に基づく新規生理活性ペプチドの発見発表者は、ゲノムマイニングを行い、新規環状ペプチドの発見を行っている。特に、 Streptomyces curacoi から 2 種類の環状ペプチド curacomycin 1) および curacozole 2)が発見された。Curacomycin は塩化トリプトファンを含む環状ヘキサペプチドであり、抗菌活性を示した。また、curacozole はオキサゾールおよびチアゾールを多数構造に含む環状ペプチドであり、顕著な細胞毒性活性を示した。また、ゲノム情報から、両化合物の生合成遺伝子クラスターの推定を行った。

21 2．ラッソペプチドの異宿主生産ラッソペプチドとは細菌のよって生産される環状ペプチドであり、N 末端側 7-9 アミノ酸残基が環状構造を形成し、C 末端側の直鎖ペプチド部分が輪の中を貫通する特異な構造を有するペプチド分子である。近年の中分子ペプチドに対する注目の高まりにより、ラッソペプチドに注目が集まっている。発表者は、ゲノムマイニングに基づいて、新規ラッソペプチドの構造決定に取り組んでおり、数々のラッソペプチドの構造決定に成功している。また、大腸菌-Sphingomonas 間で使用できるシャトルベクターを用いて、新規ラッソペプチドの異宿主生産に成功した 3)ので詳細を紹介したい。

Brevunsin の遺伝子クラスター（黒色） Sphingomonas 属細菌細胞を用いを発現用シャトルベクターに組み込む。て、新規ラッソペプチド brevunsin

の異宿主生産に成功した。参考文献 1) I. Kaweewan, H. Komaki, H. Hemmi, S. Kodani, Isolation and structure determination of new antibacterial peptide curacomycin based on genome mining. Asian J. Org. Chem., 6 (12), 1838–1844, 2017. 2) I. Kaweewan, H. Komaki, H. Hemmi, K. Hoshino, T. Hosaka, G. Isokawa, T. Oyoshi, S. Kodani Isolation and structure determination of a new cyototoxic peptide curacozole from Streptomyces curacoi based on genome-mining. J. Antibiot., 72(1), 1-7, 2019. 3) S. Kodani, H. Hemmi, Y. Miyake, I. Kaweewan, H. Nakagawa, Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranean. J. Ind. Microbiol. Biot., 45 (11), 2018.

22 医薬品分野における経済産業省の取り組み浜田盛之（経済産業省商務情報政策局生物化学産業課） [email protected]

日本では高齢化が急速に進展しており、2060 年には全人口に占める 65 歳以上人口の割合は 38%に達すると予測されている。高齢化や社会環境の変化に伴い疾患の性質も変化しており、がんや認知症といった老化に伴う疾患が大きな問題となっている。医療の課題として、患者の方々の QOL を向上させるとともに、医療費増加の抑制を図る必要がある。経済産業省では先制医療、個別化医療を推進するため、日本医療研究開発機構（AMED）を通じて、「次世代治療・診断実現のための創薬基盤技術開発事業」を実施しており、現在以下の取り組みを行っている。〇体液中マイクロ RNA 測定技術基盤開発がん細胞が分泌する血中マイクロ RNA の検出技術を確立し、低侵襲かつ早期発見が可能ながん診断技術を開発する。〇中分子創薬の基盤技術開発中分子創薬を加速する基盤技術として、中分子の構造多様性を拡大する技術および細胞膜を透過できる構造を予測する技術を開発する。〇糖鎖利用によるバイオ医薬品の高度創薬技術開発がん細胞等の疾患細胞に発現する特異的な構造を持つ糖タンパク質を高感度に探知する技術を開発し、新薬開発促進に繋がる技術基盤を整備する。〇バイオ医薬品の高度製造技術開発バイオ医薬品の大量生産を可能とする国内産の生産細胞株を樹立するとともに、世界に先駆けてバイオ医薬品の連続生産技術を開発する。〇患者層別化マーカー探索技術開発（平成 31 年度開始予定）奏効率が低く個別化医療が求められる抗がん剤等の薬剤に対して、患者を層別化可能なマーカーを開発する。

本講演では、上記研究課題の概要と経産省プロジェクトの特徴について紹介する。

23 「二次代謝物の生合成経路の悉皆的理解を目指したデータサイエンス」金谷重彦（奈良先端科学技術大学院大学・先端科学研究科データ駆動型サイエンス創造センター兼務） [email protected]

オミクスと薬用/食用の知識を統合的に扱ったプラットフォームに従ってデータベースを構築すれば、社会の最重要課題である「健康」「医薬」を課題とした情報を体系的に検討できる。そこで、メタボローム研究を中心に薬用・植物知識ベース（機能性、配合）、さらにヒト生理活性を統合的に扱うデータベース KNApSAcK Family DB (http://kanaya.naist.jp/ KNApSAcK_Family/) 1) の構築を進めている。KNApSAcK Core System には、生物種と二次代謝物の関係データ情報が整理されており、現在までに、114,238 レコードの生物種-二次代謝物の関係、二次

代謝物の総数は 51,086 種となって図 1 KNApSAcK family DB のメインウインドウ (CobWeb をクリックすると図２が表示される) いる。また、白井博士（長浜バイオ大学）が開発した代謝物の三次元グ

図２ CobWeb メインウインドウ(生物種検索のテキストボックスに Streptomyces と入力すると、この生物に関わる代謝マップがハイライトされる（ピンク色）、マップ 1 を選択すると図３が表示される。)

24 ラフマッチングアルゴリズム(COMPRIG) 2)により、Twins DB においては二次代謝物間の類似性を検索することが可能になった。現在までに、生物種、二次代謝物にかかわる 15 種のデータベースの開発を進めている。本講演では、KNApSAcK DB における代謝物と生物種の関係データベースを中心に、現在構築を進めている二次代謝生合成データベース CobWeb (http://kanaya.naist.jp/CobWeb/top.jsp)を紹介する。また、深層学習の一つであるグラフ・コンボリューション・ネットワークにより化学構造によるアルカロイド生合成経路における開始物質の予測を行ったところ、95%程度の精度で予測が可能になったので、この成果についても紹介したい。

図３代謝マップ 1、ピンク色の部分は Streptomyces で報告されている二次代謝物。（代謝物をクリックすると、代謝物が報告されている生物種の情報が得られる）

参考文献 1) Afendi FM et al., KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research, Plant Cell Physiol. 53, e1, 2012. 2) Saito M, Takemura N, Shirai T, Classif ication of ligand molecules in PDB with fast heuristics graph match algorithm COMPLIG. J. Mol. Biol. 424, 379-390, 2012.

25 全自動１細胞解析単離装置の開発および微生物研究への応用黒田俊一（大阪大学産業科学研究所・生体分子反応化学研究分野） [email protected]

我々は 10 万個以上の膨大な数の細胞群から、１細胞単位で好ましい形質を有する細胞を見出して、その細胞だけを全自動で非侵襲的に１細胞単離する「全自動１細胞解析単離装置（一般名称、ワンセルマシン）」を、大阪府立大学、神戸大学、アズワン株式会社、古河電気工業株式会社、スターライト工業株式会社との共同研究により開発した。本装置は、格子状に配置した 10~30 ミクロンのウェル（総数 10~30 万）を有するセルアレイ部分、蛍光顕微鏡部分、グラスキャピラリを駆動するマイクロマニピュレータ部分により構成される。セルソーターとの大きな相違点は、①細胞群の中から最も好ましい形質を示す細胞を１細胞単位で単離できる、②培養液中で実施可能、③化学的・物理的な細胞ストレスが極小、④Dead volume が極めて少ない、⑤貴重な細胞の回収が可能、⑥細胞接触部分がディスポーザブルなど、である 1)。これまでに、ワンセルマシンを用いることで、有用物質分泌細胞群の中から最も分泌量の多い細胞の１細胞単離して育種する有効性を示したり 2)、幹細胞群の中から最も多分化能が高い細胞を１細胞単離して育種する有効性を示したりして、コロニー形成を前提とした従来の「１コロニー育種」ではなく、「１細胞育種」を提唱してきた 3,4)。また、ヒトサイトカイン受容体とペプチドライブラリーを細胞表層で共発現させた出芽酵母を用いて、de novo アゴニストスクリーニングを迅速に行うことにも成功した 5-7)。さらに最近では、ワンセルマシンに、セルアレイ上の細胞群が発する蛍光強度変化を１細胞単位で経時的に記録する Time-lapse Single Cell Array Cytometry の機能を搭載し、その蛍光パターンに基づき１細胞単離を行い、特定のニオイ分子に応答するマウス嗅上皮由来の嗅覚受容体群を網羅的に単離することにも成功した 8)。これまでワンセルマシンの適用は、蛍光顕微鏡で容易に可視化される動物細胞、出芽酵母のみであったが、最近、我々はエマルジョン内に微生物を１細胞単位で封入し、ワンセルマシンでエマルジョン封入微生物の１細胞単離を可能にした。また、エマルジョン内は各種酵素反応を行うのに最適であることから、様々な微生物の酵素反応を介した「１細胞育種」が現実味を帯びてきている。これまで、全微生物の 99%を占めるとも言われる難培養性微生物のハンドリングは iChip などの成功例はあるものの、依然として大きな課題であったが、我々の開発したエマルジョンとワンセルマシンを併用すれば打開できる可能性がある。

26 参考文献 1) Yoshimoto N. et al., An automated system for high-throughput single cell-based breeding. Sci. Rep. 3, 1191, 2013. 2) Kida A. et al., Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level. Anal. Chem. 85, 1753-1759, 2013. 3) Yoshimoto N. and Kuroda S., Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties. J. Biosci. Bioeng. 117, 394-400, 2014. 4) Tatematsu K. and Kuroda S., Automated Single-Cell Analysis and Isolation System: A Paradigm Shift in Cell Screening Methods for Bio-medicines. Adv. Exp. Med. Biol. 1068, 7-17, 2018. 5) Yoshimoto N. et al., High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system. Sci. Rep. 4, 4242, 2014. 6) Yoshimoto N. et al., Cytokine-dependent activation of JAK-STAT pathway in Saccharomyces cerevisiae. Biotechnol. Bioeng. 113, 1796-1804, 2016. 7) Yoshimoto N. and Kuroda S., High-throughput analysis of mammalian receptor tyrosine kinase activation in yeast cells. Methods Mol. Biol. 1487, 35-52, 2017. 8) Suzuki M. et al., Deciphering the receptor repertoire encoding specific odorants by time-lapse single-cell array cytometry. Sci. Rep. 6, 19934, 2016.

27 報告第 65 回日本放線菌学会学術講演会

主催：日本放線菌学会

日時：令和元年 5 月 24 日（金） 13:00〜17:40

場所：山梨大学大村智記念学術館大村記念ホール

参加者： 51 名

プログラム

1．『糸状菌の未利用遺伝子資源にアクセスする複雑系培養法の研究』萩原大祐（筑波大学生命環境系糸状菌相互応答学講座）

2．『昆虫における腸内共生の分子基盤』菊池義智（(国研) 産業技術総合研究所北海道センター）

3．『なぜ多くの微生物は培養困難なのか？』青井議輝（広島大学統合生命科学研究科生物工学ユニット）

4．『放線菌から学んだこと』― Kitasatospora 属と Patulibacter 属の発見と分類 ― 高橋洋子（北里大学北里生命科学研究所創薬資源微生物学）

5．『県産素材を用いた食品開発とワインのティスティング』柳田藤寿（山梨大学生命環境学部地域食物科学科ワイン科学研究センター）

28 糸状菌の未利用遺伝子資源にアクセスする複雑系培養の研究萩原大祐（筑波大学・生命環境系・糸状菌相互応答学講座） [email protected]

糸状菌は多様な生理活性物質を産生し、これらの化合物が抗生物質をはじめとする医薬や農薬などに利用され、人類の健康で快適な生活に幅広く貢献している。糸状菌のゲノムにはおよそ 10,000-14,000 個の遺伝子が存在し、二次代謝産物の産生に関わる遺伝子クラスターは、約 30-70 個と推定される 1)。それに対して、単独の菌種から産生が認められる化合物の数は大きく下回っており、未利用化合物が多く残されていると考えられている。近年、糸状菌の潜在的な化合物生産能に着目し、未利用化合物およびその生合成遺伝子の解析が進み、最も解析の進んだ Aspergillus fumigatus では半数以上の遺伝子クラスターの生産化合物が明らかになってきた 2)。これらの解析は、遺伝子強制発現や異種発現といった遺伝子工学的なアプローチによりなされており、遺伝子の発現が機能発現にとって重要であることを良く示している。実験室環境の培養で、どれほどの遺伝子が発現しているのかを明らかにするため、複数の Aspergillus 属糸状菌を多様な培地で培養しトランスクリプトーム解析を行ってみると、発現が一定レベルを越えた遺伝子は、全遺伝子の 65-86%であり、二次代謝生合成遺伝子では 7.2-50%と低い割合であった。このことから、未利用化合物の多くは実験室環境で発現できないために産生されず解析が進んでいないと推測される。したがって、これらの二次代謝遺伝子にアクセスするためには、発現を促す工夫が必要となる。微生物が実際に生息する環境は、実験室で遭遇するような豊富な栄養を含んだ均質な培地環境や、他の生物が完全に存在しない純粋培養の環境から大きくかけ離れている。そこで、より実環境に近い条件で培養することにより、実験室環境では発現が確認されていない遺伝子の発現を誘導できるのではないかと考えた。現在私たちのグループでは、物性的、化学的に複雑な土壌を用いた培養（土壌培養法）、および複数の微生物を混合した培養（混合培養法）により、糸状菌の未利用遺伝子の発現誘導を検討している。土壌培養法においては、モデル糸状菌である Aspergillus nidulans を用いて、最適な培養法を確立し、トランスクリプトーム解析により未利用遺伝子の発現誘導の可否を検証した。また、寒天培地とは異なる培養性状が確認されるなど、本培養法により糸状菌の真の生態に迫ることが可能になると期待される。混合培養法では、A. nidulans と A. fumigatus の二者を培養することにより、単独培養ではみられない化合物生産の誘導が認められた。したがって、従来の単独種による培養では確認されなかった化合物の生産

29 が、混合培養法により解析可能になると考えられ、広範な糸状菌種に適用することで新たな遺伝子資源にアクセスできることが期待される。今後、これらの複雑な培養法の解析をさらに進めることにより、糸状菌の未利用遺伝子や有用物質の発見に繋げるとともに、その現象を支える分子機構を明らかにし、包括的な理解を目指していく。さらに、糸状菌が産生する生理活性物質の生態における機能解析を通じて、実環境における糸状菌の真の生態に迫ることができると期待している。

参考文献 1) Inglis DO et al., BMC Microbiol. 13: 91, 2013. 2) Keller NP, Nat. Rev. Microbiol. 17: 167, 2019.

昆虫における腸内共生の分子基盤菊池義智（（国研）産業技術総合研究所・北海道センター） [email protected]

昆虫は 100 万種以上が知られる陸上最大の生物グループであり、その半数以上が体内に何らかの共生微生物を持つと推定されている。これら共生微生物は宿主昆虫の栄養代謝において必須の役割を果たしており、母子間伝達によって次世代に連綿と受け継がれる。昆虫共生微生物の多くは宿主体内環境に高度に適応しているために培養が難しく、これにより共生の遺伝的基盤に関する研究が大きく立ち遅れてきた。近年のオミクス技術の革新はこのような昆虫共生微生物研究における停滞感を打破するに十分であったが、共生の仕組みを総合的に理解するためには未だ埋めるべき大きな溝がある。最近我々は、農作物の重要害虫であるホソヘリカメムシ（Riptortus pedestris）が非常にユニークな内部共生系を発達させており、何より共生微生物が培養可能であることを発見した。ホソヘリカメムシは消化管に多数の袋状組織（盲嚢）を発達させており、その中に Burkholderia 属の共生細菌を保持している。他の多くの昆虫とは異なり、ホソヘリカメムシは Burkholderia 共生細菌を母子間伝播することなく毎世代土壌中から獲得する。Burkholderia 共生細菌は、カメムシ消化管はもとより土壌からも容易に単離・培養することが可能であり、また遺伝子組換えにより特定の遺伝子を knock out/knock in することが容易にできる。本講演では、ホソヘリカメムシと Burkholderia を用いたこれまでの研究展開について、特に（１）共生特異性に関わる分子基盤、および（２）昆虫腸内細菌が担う新機能“農薬分解”に焦点を絞り、最新データも交えてご紹介する。

30 参考文献 1) Kikuchi, Y. Endosymbiotic bacteria in insects: their diversity and culturability. Microbes Environ. 24: 195-204, 2009. 2) Itoh, H., K. Tago, M. Hayatsu, and Kikuchi Y. Detoxifying symbiosis: microbe- mediated detoxification of phytotoxins and pesticides in insects. Nat. Prod. Rep. 35: 434-454, 2018.

なぜ多くの微生物は培養困難なのか？青井議輝（広島大学大学院・統合生命科学研究科・生物工学ユニット） [email protected]

微生物のほとんどは培養困難であることが知られており、未解明・未利用のまま広大なフロンティアが残されている 1)。近年の培養非依存的な網羅的解析手法の著しい進展により広大な未知領域の存在が明らかになったが、「鍵」となる微生物が培養できないため本質的な理解（因果関係など）や利用拡大に制限がかけられている状況でもある。そこで、多くの微生物は培養困難な普遍的な理由やメカニズムを解明できれば、現状では有効技術のない難培養性の未知微生物を培養化するための画期的な新戦略を導き出せるかもしれない。しかし，難培養性を説明可能な普遍的な理由は全く明らかになっていない。また、通常の培養法で容易に獲得（分離培養）できる微生物を解析しても「なぜ培養できないのか」という問いに対する答えには到達しないというジレンマが存在する。さらに、従来法に替わる画期的な分離培養手法もほとんど登場していない。【新しい分離培養手法】そこで、我々の研究グループでは、新規分離培養手法を開発してその有効性を実証しつつ、さらにそれらの手法を通じて得られた難培養性微生物を用いて未知なる増殖制御機構の解明に取り組んでいる。以下にその一部を挙げる。１）微生物の罠 2)：ナノメートルオーダーの構造体をデザインすることで、現場に設置するだけで自動的に複数の菌株をサンプリング・分離・培養する全く新しいタイプの分離培養手法。本手法は、人為的に微生物を分離・植菌するのではなく、「微生物自身が能動的に分離し、隔離した状態で増殖する」という点で既存の方法論に対して大きく異なる。２）対象微生物の選択的分離 3)：セルソーターを用いて形態学的な情報に基づいて対象微生物を生きたまま選択的に分離する方法。３）in situ 培養 4)：微生物は通過しない膜を用いた独立した培養チャンバーで構成された培養デバイスを用いた、実環境を模擬する培養手法（in situ 培養法）。

31 ４）超高密度植菌法：培養初期の菌体密度を従来の方法の１万倍以上に高めることができ、培養期間中お互いに混在しないで分離した状態で増殖させる方法。培地条件は同一にもかかわらず従来法の 10 倍程度高い培養効率（コロニー形成率）を示すことが判明した。【難培養性微生物とは何か】なぜ多くの微生物はなぜ培養できないのか？そもそも普遍的な要因が存在するかどうかも定かではない。つまり、常識的には、多くの微生物が培養できない理由は「培地組成など培養条件が適合しない」こと（だけ）が原因ではないかと考えられる傾向にある。しかし我々は、このような「培養条件の不適合性」以外に、まだ十分に検討されていない普遍的な要因があり得ると仮定して検討した結果、実際に以下のような性質を見出している。性質１）休眠と覚醒：多くの環境中の微生物は好ましくない条件下では容易に非増殖状態（休眠状態）に移行する。環境状況が好転しても休眠状態からは容易に脱却せず、そこから脱却するためには、シグナル様物質（覚醒因子）が必要である。そしてそれは増殖状態の特定の微生物（異種・同種）から分泌される。性質２）多くの難培養性微生物（未培養微生物）は、自身が産出する代謝物（代謝副産物）に増殖を顕著に阻害され、コロニー形成が不可能であるなど従来法では容易に分離できない。また上記の１）および２）の性質を両方併せ持つような微生物種は極めて分離培養が困難である。実際に、上記に挙げた新規培養手法で得られた新規性の高い微生物の多くは、上記の性質をどちらか、または両方持っていることが判明した 5,6)。【まとめ】これまでに得られた結果は、「個々の微生物種の適合性」だけではなく、普遍的に環境微生物の難培養性をコントロールするメカニズムが存在することを示唆している。また、微生物の「難培養性」について本質的な理解を得ること，すなわち増殖という微生物の活動の本来根幹となる部分を理解することに直結する。さらに、分離培養手法の革新や培養可能な微生物の拡充，そして環境微生物の「難培養性」を解明することにより、直接利用できるバイオリソースの大幅な拡大が見込まれることも期待できる。

参考文献 1) Rinke et al. Nature 2013. 2) Tandgan et al., PLOS ONE 2014. 3) Fujitani et al., Enviorn. Microbiol., 2014. 4) Aoi et al., Manuals of Environmental Microbiology 4th edition, 2016. 5) Jung et al., submitted. 6) Jung et al., submitted.

32 放線菌から学んだこと —Kitasatospora 属と Putalibacter 属の発見と分類— 高橋洋子（北里大学北里生命科学研究所・創薬資源微生物学） [email protected]

これまでに、様々な方法で多くの放線菌を分離してきた。その培養液の中から北里生命研大村創薬グループによって類縁体も含めて約 280 の新規化合物が発見された。一方、これらの分離株から、1 科、16 属、71 種の新分類群を提唱してきた。今回は、その中の Kitasatospora 属と Putalibacter 属の発見に至る経緯と分類について述べる。

1. 新属 Kitasatospora 属の提唱 Kitasatospora 属１）は、寒天培地上で培養したコロニーの形態は Streptomyces 属と同様に気菌糸に長い胞子の連鎖を形成する。しかし、化学分類の重要な指標の一つである細胞壁ジアミノ酸として LL-ジアミノピメリン酸（DAP）と meso-DAP の両方を含んでいる。それまで報告された属は、どちらか一方を含むものであり、コンタミネーション説なども出されたが、K. setae KM-6054T を基準種として新属を提唱した（1982 年）。図 1 に示したように、細胞壁に LL-DAP を含む胞子から meso-DAP をもつ菌糸が伸長する。その後、この菌糸が LL-DAP をもつ胞子を新たに形成し、ここからさらに meso-DAP を含む菌糸が伸長する。このサイクルを繰り返す。この実験によって、1 種類の放線菌であることを証明した。その後、本属の新種の報図 1. K. setae KM-6054T の液体培養おける告が相次いだが、1992 年に形態と DAP 異性型の経時変化 Streptomyces 属の定義が LL-と meso-DAP の両方を含む場合もあると変更され、本属が Streptomyces 属に移行された。そして、1997 年には 16S rDNA の塩基配列解析によって、単独の属として元に戻された。現在、承認名として 31 種報告されている。このような変遷の中でも、本属の

33 特徴と K. setae KM-6054T を用いて行った形態分化の過程や細胞の化学組成が否定されたことはなかった。

2. Putalibacter 属の発見と新科、新属の提唱自然環境と実験室環境との隔たりはどれほどのものか想像できない。環境中に生息している微生物種の 99%は未だ分離できていないと言われる。分離プレート上に様々なコロニーが出現してくるが、その中に、同じコロニーが多数出現してくることがある。この菌株は、他の菌株に何らかの影響を与えている可能性があると考え、この培養液を分離用寒天培地に添加してみた。無添加では出現しない菌株が多数得られた。この培養液中の菌株増加物質はスーパーオキシドジスムターゼ 2) - (SOD)であった。一般的な寒天培地から活性酸素（O2 ）が発生しており、カタラーゼの同時添加で、さらにコロニー数が増加した 3) 。この方法で分離された菌株から、新科 Patulibacteraceae、新属 Patulibacter minatonensis KV-614T4)を筆頭に、1 科、3 属、 8 種の新分類群を提唱した。P. minatonensis KV-614T は、G+C 含量 72 mol% で長い鞭毛を有する桿菌である。16S rDNA 塩基配列で Blast 検索すると土壌からクローンでのみ確認されているものや、希釈培地で 3 ヶ月間培養して得られた菌株等がヒットする。また、Patulibacter 属近縁菌の特異プライマーを作成し、様々な環境の土壌試料から PCR 検出を行ったところ 72%（31/43 試料）に同一バンドが検出され、同様の菌株は広く環境中に存在するにも関わらず分離されていないことが示唆された 5)。 99% の中のごく僅かではあるがこれまで分離されなかった菌株に巡り合った。しかし、これらの菌株が、環境中でも SOD 生産株の助けを受けているのかどうかは不明である。

3. 放線菌の分離と分類に期待すること分類は、人間が何らかの指標を設けて生物をグルーピングすることであり、そこに当てはまらないものが出現する。放線菌自体が、原核生物であるが複雑な形態分化を起こす境界線上にある微生物群と言える。また、形態観察は寒天培養で行われるが、培養条件によってばらつきがでる。これに客観性を与えるために化学分類が導入された。しかし、解析には液体培養菌体が用いられるという矛盾も抱えてきた。そして今は、特定の遺伝子の塩基配列による分類が優勢となり、そのことによって多くの属が誕生している。ゲノム情報が急速に増加している今こそ、表現型を丁寧に記載し、これらの表現系とゲノム情報とを紐づけする地道な作業が大切である。このことが、分離や分類における我々の課題に有益な情報を与えてくれると同時に、生物種の特徴がより反映された分類に発展するのではないかと考える。

34 参考文献 1) Takahashi Y. J. Antibiot., 70: 506-513, 2017. 2) Takahashi Y. et al. J. Gen. Appl. Microbiol., 49: 263-266, 2003. 3) Nakashima T., et al. J. Biosci. Bioeng., 110: 304-307, 2010. 4) Takahashi Y. et al. Int. J. Syst. Evol. Microbiol., 56: 401-406, 2006. 5) Seki T. et al. J. Antibiot., 68: 763-766, 2015.

県産素材を用いた食品開発とワインのティスティング柳田藤寿（山梨大学生命環境学部地域食物科学科・ワイン科学研究センター） [email protected]

日本のワインについて日本には、約２８０のワイナリーがあり、そのうち山梨県には、約 80 のワイナリーがあり全国一のワイン生産量の県です。近年、日本のアルコール飲料に関して、消費量が右肩上がりの日本ワインの最新の情報とワイン用ブドウ（甲州ブドウやマスカット・ベーリーA ぶどう）の歴史について、講演します。また実際に白、赤ワインのティスティングも行います。また、演者は以下のワインや飲料などの商品開発も行っていますので、これらの内容に関しても講演します。世界初の海洋酵母ワインの研究海洋酵母は、三共製薬会社（現第一三共）が新薬の開発のために海洋環境から分離した酵母で、この酵母を用いて 2000 年「サッポロワイン（株）」から「海の酵母ワイン」を開発しました。幻の湖-富士六湖（赤池）からのワイン開発赤池は、6〜７年に一度、現れる富士山麓の「幻の湖」で、湖水から発酵力の強い酵母を分離しました。この中から優良酵母を選抜し 2014 年に「白百合醸造（株）」から「赤池幻酵母ワイン」を、を開発しました。甲府市における発酵性酵母の分離とスパークリングワインへの応用甲府市は、2019 年に開府５００年を迎えました。そこで甲府市との共同研究で武田神社のお堀の水を分離源とした優良ワイン酵母の探索を行いました。この中から優良株を選抜し、地域特産スパークリングワインの製品化を行い、2018 年「甲府スパークリング甲州 2017」の名前で（株）サドヤから発売し予約で完売するなど、好評でした。大豆で作った飲むヨーグルトの研究 2010 年に飲料用大豆「すずさやか」を使用し、北杜市の「尾白川の水」で仕込み、「大豆丸ごと製法」のため食物繊維も多く含まれており、ワイン酵母で発酵を行ったことから、大豆臭の少ない大豆で作った飲むヨーグルト飲料を「白州屋まめ吉」と開発しました。

35 食べるブドウジュースの研究 2016 年に、ブドウを種も皮も実までまるごと使った「食べるブドウジュース」の飲料を「山梨 Made」と開発しました。一般的なぶどうジュースではとれないビタミン E やオレイン酸やリノール酸が含まれていて、総プロアントシアニジンやレスベラトロールも多く含まれています。

36 日本放線菌学会賛助会員

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）生物資源研究所合同酒精（株）酵素医薬品研究所味の素(株) イノベーション研究所トヨタ紡織株式会社基礎研究所富士シリシア化学（株）チーム未来グループ

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