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Roles and Quality Management of Culture Collection in Genomic Roles and Quality Management of Culture Collections in Genomic Era 21 November 2018 WDCM GCM2.0 Workshop 127 Ken-ichiro Suzuki Department of Fermentation Science Faculty of Applied Bioscience Tokyo University of Agriculture (TUA) 鈴木健一朗 東京農業大学応用生物科学部醸造科学科 Relationship between Culture Collection and Taxonomy (Summary of Today’s Talk) Taxonomy Scientific background Providing Type strains Academic Community Social Infrastructure Culture Collections and the Network 21/11/2018 Whole Genome Sequences – Summary of Today’s Talk – • Research purpose – Application – Taxonomic comparison without transfer of strains – Taxonomy for In silico hybridization • Quality management of collection – Trace the transfer at strain level – Identity confirmation with the depositor – Identity confirmation with other culture collections 21/11/2018 Whole Genome Sequence Analysis for Taxonomic Studies Courtesy of NBRC Culture Collection 21/11/2018 Relationship of 16s rRNA homology and DNA-DNA reassociation values DNA-DNA reassociation (%) 0 10 20 30 40 50 60 70 80 90 100 100 × △△ △ ▲▽ ×× × ▽ × ×△ △▽× △ ▽ △ ▼ × ▽ ▽ × ▼ 99 × ▼× × × ◎▽ 98 ◎◎ ◎ ××× ◎ ◎◎ 97 × ◎ ▼ ◎ ◎ ▼ ◎ 96 × ××× × × ▼ 95 DNA threshold value 94 for species delineation 16S rRNA homology homology (%) rRNA 16S 93 × 92 × × × : Membrane filter method 91 △ ▲ ▽ ▼ : Renaturation method Stackebrandt & Goebel, 1994 ◎ : S1 nuclease method 21/11/2018 Principle of DNA-DNA Hybridization One of the most essential methods to elucidate the species level Identification. Fragmen- Heat tation denatured Labeling Hybridization reaction (RI, fluorescent) Organism A (Reference) Heat Ratio = Index (ds) (ss) Immobilized Washing denatured Organism B 21/11/2018 Judgement of the Results of DNA-DNA Hybridization (%)100 A: Species 90 B: Subspecies DNA B C: Other subspecies 80 A in the same species - DNA Relatedness DNA 70 D: Other species C in different species 60 (J. L. Johnson 1973, 1984) 50 40 D The phylogenetic definition of 30 u species generally would include strains with 20 approximately 70% or greater 10 DNA-DNA relatedness and 0 with 5°C or less DTm. 21/11/2018 (Wayne, et al. 1987) Definition of Bacterial species Based on DDH The species is the basic unit of bacterial taxonomy and is defined as a group of strains, including the type strain, sharing 70% or greater DNA-DNA relatedness with 5ºC or less ΔTm (Vandamme et al. 1996, Wayne et al. 1987). However, DNA-DNA hybridization (DDH) is … There are several different methods. There are many parameters such as salt concentration, temperature, etc. It is easily affected by experimental error. It is required high-skilled techniques. There are times that the values may be different between the papers. 21/11/2018 In silico DNA-DNA Hybridization • Various methods have been developed for bacterial taxonomy using genome sequences. • The relationship between in silico DDH and traditional DDH was reported. • Advantage of In silico DDH is based on the absolute data of WGS The average nucleotide identity (ANI) Konstantinidis & Tiedje, Proc Natl Acad Sci USA 2005, 102, 2567–2572 Goris et al. Int J Syst Evol Microbiol, 2007, 57, 81–91 Richter & Rosselló-Móra, Proc Natl Acad Sci USA, 2009, 106, 19126-19131. 95-96% ANI corresponds to 60-70% DDH. Genome-To-Genome Distance Calculator (GGDC) Auch et al., Stand Gen Sci, 2010, 2, 117-134 Auch et al., Stand Gen Sci, 2010, 2, 142-148 0.0412 GGDC formula (2) value corresponds to 70% DDH. DNA maximal unique matches index (MUMi) Deloger et al., J Bacterial 2009, 191, 91-99 0.33±0.03 MUMi corresponds to 95%±0.5% ANI. 21/11/2018 Case 1: Proposal of New Species Based on DDH - Taxonomic Study of Novel Strains Belonging to the Family Demequinaceae Isolated Using SPPY Agar Studied by Dr. Moriyuki Hamada, NBRC, Skerman Award Winner 2017 21/11/2018 NBRC Culture Collection 21/11/2018 Phylogenetic Tree of the Isolates Based on 16S rRNA Gene Sequences 96 HI12-100 100 HI12-104 HI12-140 0.01 Knuc 61 HI12-128 100 HI12-44 HI12-143 71 HI12-121 85 Lysinimicrobium cluster 98 HI12-114 92 HI12-135 61 Lysinimicrobium mangrovi NBRC 105856T (AB639012) 91 HI12-122 HI12-123 61 HI12-45 HI12-111 63 Demequina flava NBRC 105854T (AB674956) 71 64 Demequina globuliformis NBRC 106266T (AB639018) 95 Demequina sediminicola NBRC 105855T (AB674957) 62 HI12-62 68 HI12-125 77 HI12-149 100 HI12-106 82 96 Demequina salsinemoris NBRC 105323T (AB639017) Demequina cluster HI12-66 100 HI12-147 89 HI12-71 Demequina aestuarii NBRC 106260T (AB639015) T 68 100 Demequina aurantiaca NBRC 106265 (AB639020) Demequina oxidasica NBRC 106264T (AB639019) 63 Demequina lutea NBRC 106155T (AB639016) Actinotalea fermentans NBRC 105374T (AB639014) 89 Cellulomonas flavigena DSM 20109T (X83799) 73 Cellulomonas cellasea DSM 20118T (X83804) 66 Cellulomonas bogoriensis 69B4T (X92152) Oerskovia turbata DSM 20577T (X83806) 100 Oerskovia enterophila DSM 43852T (X83807) Sanguibacter marinus 1-19T (AJ783958) 93 Sanguibacter keddieii ST-74T (X79450) 21/11/2018 Brevibacterium linens DSM 20425T (X77451) Polyphasic Approach by Chemotaxonomy Characteristic 13 isolates Lysinimicrobium 7 isolates Demequina (Lysinimicrobium spp.) (Demequina spp.) Peptidoglycan type A4α A4α A4β A4β Diamino acid L-Lys L-Lys L-Orn L-Orn Interpeptide bridge D-Glu or D-Glu L-Ser–D-Glu or Ser–D-Asp or D-Ser–D-Glu D-Glu Ser–D-Glu Major menaquinone DMK-9(H4) DMK-9(H4) DMK-9(H4) DMK-9(H4) Major fatty acids ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, ai-C15:0, ai-C17:0, C16:0 C16:0 C16:0 C16:0 Polar lipids§ DPG, PG, PI, PIMs, DPG, PG, PI, PIMs, DPG, PG, PI, DPG, PG, PI PL PL PIMs, PL Genome size 2.68–3.23 Mbp 2.97Mbp 2.50–3.35 Mbp 2.46–3.21 Mbp DNA G+C content 71.4–72.4 71.8 68.9–71.1 62.7–70.2 §DPG, Diphosphatidylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIMs, phosphatidylinositol-mannosides; PL, unknown polar lipid. 21/11/2018 ANIb Values and 16S rRNA Gene Sequence Similarities among the 13 Isolates and L. mangrovi NBRC 105856 Proposal of 9 New Species for 13 Isolates 16S rRNA Gene Sequence Similarity ANIb 21/11/2018 Hamada et al. 2015 Comparison of the Dendrograms by 16S, MLSA and ANI HI12-140 0.01 0.02 87 0.02 HI12-140 96 HI12-100 100 HI12-104 HI12-104 100 HI12-104 HI12-100 100 HI12-100 HI12-140 HI12-121 HI12-121 61 HI12-128 HI12-114 100 HI12-114 100 HI12-44 79 HI12-135 HI12-135 HI12-143 HI12-128 100 100 HI12-123 71 HI12-121 52 85 HI12-44 HI12-45 98 HI12-114 95 92 L. mangrovi HI12-135 HI12-122 61 HI12-143 L. mangrovi 100 HI12-44 91 HI12-123 HI12-122 HI12-128 99 100 HI12-45 HI12-123 HI12-143 HI12-122 61 HI12-45 L. mangrovi HI12-111 HI12-111 HI12-111 87 D. flava 63 D. flava D. salsinemoris 100 D. globuliformis 71 64 D. globuliformis 96 HI12-106 D. sediminicola 81 HI12-66 95 D. sediminicola 100 HI12-62 62 HI12-62 D. flava HI12-125 68 HI12-125 100 100 D. globuliformis 77 HI12-149 HI12-149 D. sediminicola HI12-147 HI12-106 100 HI12-62 66 HI12-71 82 96 D. salsinemoris HI12-125 83 D. aestuarii HI12-66 100 HI12-149 D. salsinemoris 100 HI12-147 100 HI12-71 89 HI12-71 HI12-106 100 HI12-147 D. aestuarii HI12-66 D. aestuarii 68 100 D. aurantiaca D. aurantiaca D. aurantiaca 100 D. oxidasica D. oxidasica D. oxidasica 63 D. lutea D. lutea D. lutea 16S rRNA gene MLSA ANI (Concatenated amino-acid sequences of atpD, gyrB, recA, rpoB and trpB genes) 21/11/2018 Conclusion • 20 Strains which phylogenetically related to members of the family Demequinaceae were isolated from Iriomote Island, Japan, using the SPPY agar • Of them, 13 strains formed a monophyletic cluster with Lysinimicrobium mangrovi and remaining 7 strains were included in Demequina cluster • Major chemotaxonomic characteristics of the strains corresponded to those of the genera Lysinimicrobium and Demequina, respectively • Results of in silico DNA-DNA hybridization suggested that 20 strains should be assigned to 9 new species of the genus Lysinimicrobium and 7 new species of the genus Demequina, respectively 21/11/2018 Case 2. Speciation of the Genus Nocardia using Whole Genome Sequences The genus Nocardia is a large genus of actinomycetes. The species of the genus are known not only as human/animal pathogen but also artificial organic Dr. Tomohiko compound decomposer and producers of various Tamura bioactive compounds. NBRC Nocardia alba NBRC 108234 Nocardia caishijiensis NBRC 108228 Nocardia harenae NBRC 108248 21/11/2018 16S rRNA gene sequence ANI 98 Nocardia inohanensis IFM 0092 T (AB092560) Nocardia asteroides NBRC 15531 0.01 Nocardia yamanashiensis IFM 0265 T (AB092561) Nocardia salmonicida NBRC 13393 Nocardia niigatensis DSM 44670 T (DQ659910) Nocardia soli NBRC 100376 Nocardia cummidelens NBRC 100378 Nocardia concava IFM 0354 T (AB126880) Nocardia alba NBRC 108234 Nocardia uniformis DSM 43136 T (AF430044) Nocardia ignorata NBRC 108230 Nocardia otitidiscaviarum DSM 43242 T (AF430067) Nocardia coubleae NBRC 108252 Nocardia crassostreae JCM 10500 T (AF430049) Nocardia caishijiensis NBRC 108228 Nocardia nova JCM 6044 T (AF430028) Nocardia thailandica NBRC 100428 Nocardia pseudobrasiliensis DSM 44290 T (AF430042) Nocardia harenae NBRC 108248 Nocardia miyunensis 117 T (AY639901) Nocardia cyriacigeorgica NBRC 100375 84 Nocardia jiangxiensis 43401 T (AY639902) Nocardia jinanensis NBRC 108249 Nocardia terpenica IFM 0706 T (AB201298) Nocardia testacea NBRC 100365 Nocardia mexicana DSM 44952 T (FR733723) Nocardia grenadensis NBRC 108939 Nocardia anaemiae IFM 0323 T (AB162801) Nocardia sienata NBRC 100364 Nocardia pseudovaccinii DSM 43406 T (AF430046) Nocardia rhamnosiphila NBRC 108938 Nocardia vinacea MK703-102F1 T (AB024312) "Nocardia fusca" NBRC 14340 Nocardia speluncae NBRC 108251 Nocardia caishijiensis F829 T (AF459443) Nocardia flavorosea NBRC 108225 Nocardia alba YIM 30243 T (AY222321) Nocardia carnea NBRC 14403 Nocardia jejuensis N3-2 T (AY964666) Nocardia paucivorans NBRC 100373 99 Nocardia ignorata DSM 44496 T (AJ303008) Nocardia brevicatena NBRC 12119 Nocardia coubleae OFNN11 T (DQ235688) Nocardia sp.
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