Limnochorda pilosa gen. nov., sp nov., a moderately thermophilic, facultatively anaerobic, pleomorphic bacterium and Title proposal of Limnochordaceae fam. nov., Limnochordales ord. nov and Limnochordia classis nov in the

Author(s) Watanabe, Miho; Kojima, Hisaya; Fukui, Manabu

International journal of systematic and evolutionary microbiology, 65, 2378-2384 Citation https://doi.org/10.1099/ijs.0.000267

Issue Date 2015-08

Doc URL http://hdl.handle.net/2115/62587

Type article (author version)

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Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP International Journal of Systematic and Evolutionary Microbiology Supplementary material

Limnochorda pilosa gen. nov., sp. nov., a moderately thermophilic, facultative anaerobic pleomorphic bacterium and proposal of Limnochordaceae fam. nov., Limnochordales ord. nov. and Limnochordia classis nov. in the phylum Firmicutes

Miho Watanabe, Hisaya Kojima, Manabu Fukui

Corresponding author: Miho Watanabe The Institute of Low Temperature Science, Hokkaido University, Nishi 8, Kita 19, Kita-ku Sapporo, Hokkaido 060-0819, Japan.

e-mail: [email protected] Supplementary Figure S1: The culturing procedure of strain HC45T.

Supplementary Figure S2: Phase-contrast micrograph showing -like structure of the strain HC45T (arrow) grown on NaCl-R2A liquid medium for a week.

Supplementary Figure S3: Maximum-likelihood tree based on 16S rRNA gene sequences of the strain HC45T, related environmental sequences and representatives from all classes in the phylum Firmicutes. This is based on a comparison of 1369-1449 nucleotides. Bootstrap values (percentages of 1000 replications) only 50% or more are shown at nodes.

Supplementary Figure S4: Neighbour-joining tree based on 16S rRNA gene sequences of the strain HC45T, related environmental sequences and representatives from all classes in the phylum Firmicutes. This phylogenetic tree is based on a comparison of 1369-1449 nucleotides. Bootstrap values (percentages of 1000 replications) only 50% or more are shown at nodes.

Supplemetary Figure S5: Minimum-evolution tree based on 16S rRNA gene sequences of the strain HC45T, related environmental sequences and representatives from all classes in the phylum Firmicutes. This phylogenetic tree is based on a comparison of 1369-1449 nucleotides. Bootstrap values (percentages of 1000 replications) only 50% or more are shown at nodes. Supplementary Figure S1: The culturing procedure of strain HC45T.

First enrichment culture basal medium + 2% cyclohexane Sediment of Lake Harutori

Subcultuing three times with same medium

Heating 10 mM (80ﾟC, 15 min.) fumarate

2- H2, CO2 + SO4

10 mM glucose

Isolation: twice extinction dilution

10 mM glucose 1 g l-1 yeast extract 0.1 g l-1 peptone Supplementary Figure S2: : Phase-contrast micrograph showing endospore-like structure of the strain HC45T. T 99 Thermovorax subterraneus 70B (EU370564) Supplementary Figure S3: 97 Caldanaerovirga acetigignens DSM 18802T (EF530069) 99 Thermovenabulum ferriorganovorum Z-9801T (AY033493) ML tree based on 16S rRNA Tepidanaerobacter syntrophicus JLT (AB106353) degensii KC4T (NR075034) gene sequences 99 DSM 19636T (EF554597) 79 Caldanaerobacter subterraneus 2707 T (EF554599) 99 Caldanaerobacter subterraneus subsp. tengcongensis MB4T (NR074701) Caldanaerobacter uzonensis K67T (NR115985) 96 Thermoanaerobacter italicus Ab9T (AJ250846) T , 97 Thermoanaerobacter wiegelii DSM 10319 (X92513) 99 Thermoanaerobacter sulfurophilus DSM 11584T (Y16940) Thermoanaeromonas toyohensis ToBET (AB062280) Thermacetogenium phaeum PBT (AB573433) T 62 Carboxydothermus pertinax Ug1 (AB573433) T 99 Carboxydothermus ferrireducens DSM 11255 (U76363) T 88 Carboxydothermus hydrogenoformans Z-2901 (NR074395) glycerini DSM 11254T (U82327) T 99 Moorella humiferrea DSM 23265 (GQ872425) Syntrophothermus lipocalidus TGB-C1T (AB021305) Clostridia, Clostridiales, T 99 Syntrophomonas sapovorans OM (AF022249) T Syntrophomonadaceae 99 Syntrophomonas wolfei subsp. wolfei DSM 2245 (NR074750)

93 Uncultured bacterium clone AKAU3574 (DQ125579) 68 Uncultured bacterium clone PW185 (GQ402678) 99 Uncultured bacterium clone HAW-R60-B-745d-BD (FN436105) Class/ Order OPB54 57 Hydrogenispora ethanolica LX-BT (AB669474) 99 Uncultured clone OPB54 (AF027087) Uncultured bacterium clone b19-147 (JX576081) Sporomusa sphaeroides DSM 2875T (AJ279801) T Negativicutes 99 Thermosinus carboxydivorans DSM 14886 (NR117169) Selenomonas sputigena ATCC 33510T (D89882) Desulfotomaculum acetoxidans 5575T (NR027608) 70 T 64 Desulfotomaculum nigrificans DSM574 (X62176) Thermincola carboxydiphila 2204T (AY603000) Cryptanaerobacter phenolicus DSM15808T (AY327251) Heliobacterium chlorum DSM 3682T (M11212) Clostridia, Clostridiales, 99 T 99 Heliobacillus mobilis 6 (AB100835) /Heliobacteriaceae Heliorestis daurensis BT-H1T (AF079102) T 75 99 Desulfitobacterium hafniense DSM 10664 (NR074996) Desulfitobacterium chlororespirans DSM 11544T (U68528) T 99 Desulfosporosinus orientis DSM 765 (Y11570) T 85 Desulfosporosinus auripigmenti DSM 13351 (AJ493051) Thermolithobacter carboxydivorans DSM 7242T (DQ095862) T Thermolithobacteria 99 Thermolithobacter ferrireducens JW/KA-2 (AF282254) Hydrogenibacillus schlegelii DSM 2000T (Z26934) Calditerricola yamamurae YMO722T (AB308475) T 99 Thermoactinomyces vulgaris DSM 43016 (AF138739) Planifilum fulgidum 500275T (AB088362) Caldalkalibacillus thermarum HA6T (AY753654) T 99 Erysipelothrix inopinata MF-EP02 (AJ550617) Erysipelothrix rhusiopathiae ATCC 19414T (AB034200) Erysipelotrichia/Bacilli Brevibacillus brevis ATCC 8249T (AB271756) T 88 Lysinibacillus boronitolerans DSM 17140 (AB199591) 72 Marinibacillus marinus 581T (AJ237708) 87 Bacillus subtilis DSM 10T (AJ276351) Pullulanibacillus naganoensis DSM 10191T (AB021193) Virgibacillus pantothenticus B0018T (D16275) T 62 Thermaerobacter subterraneus C21 (AF343566) 55 Thermaerobacter litoralis KW1T (AY936496) 55 Thermaerobacter nagasakiensis DSM14512T (AB061441) 99 Thermaerobacter marianensis DSM12885T (AB011495) Thermaerobacter composti Ni80T (AB454087) Sulfobacillus acidophilus DSM 10332T (NR074758) T 99 Sulfobacillus thermosulfidooxidans BC1 (U75648) 99 Sulfobacillus sibiricus N1T (NR042730) Symbiobacterium ostreiconchae KY38T (AB361629) 65 Symbiobacterium thermophilum IAM 14863T (NR075044) 65 'Symbiobacterium toebii' SC-1T (AF190460) 99 Symbiobacterium turbinis KY46T (AB455238) 95 Symbiobacterium terraclitae KA13T (AB455239) Caldinitratiruptor microaerophilus CA62NT (GQ405534) 67 Uncultured bacterium, clone SMG130 (AM930312) Uncultured bacterium, clone SMQ31 (AM930328) 99 Uncultured bacterium, clone SMG3 (AM930272) 67 Uncultured bacterium, clone SMQ8 (AM93322) Limnochordia 98 Uncultured compost bacterium, clone FS1639 (FN667155) Limnochorda pilosa strain HC45T (AB992259) classis. nov. 91 Uncultured compost bacterium, clone FS1689 (FN667168) 95 Chloroflexus aurantiacus J-10-fl (D38365) 79 ‘Thermus kawarayensis’ KW11T (NR112160) Thermotoga naphthophila RKU-10T (NR_074952) T 99 Streptomyces albosporeus NBRC 15386 (AB184637) 86 Streptomyces exfoliatus NBRC 13475T (AB184868) 54 79 Gaiella occulta F2-233 T (JF423906) Other phyla Rubrobacter xylanophilus DSM 9941T (NR074552) T 99 Leptospirillum ferriphilum DSM 14647 (AF356829) Nitrospira moscoviensis DSM 10035T (X82558) T 57 Desulfobacca acetatoxidans DSM11109 (AF002671) 83 Escherichia coli ATCC 11775T (X80725) 55 Geobacter metallireducens DSM 7210T (L07834)

0.02 T 78 Virgibacillus pantothenticus B0018 (D16275) 54 Bacillus subtilis DSM 10T (AJ276351) Supplementary Figure S4: 99 Pullulanibacillus naganoensis DSM 10191T (AB021193) NJ tree based on 16S rRNA T Bacilli 1 87 Lysinibacillus boronitolerans DSM 17140 (AB199591) 64 Marinibacillus marinus 581T (AJ237708) gene sequences 55 Brevibacillus brevis ATCC 8249T (AB271756) Erysipelothrix inopinata MF-EP02T (AJ550617) Erysipelotrichia 99 Erysipelothrix rhusiopathiae ATCC 19414T (AB034200) Selenomonas sputigena ATCC 33510T (D89882) T 99 Thermosinus carboxydivorans DSM 14886 (NR117169) Negativicutes 60 Sporomusa sphaeroides DSM 2875T (AJ279801) T 75 Desulfotomaculum acetoxidans 5575 (NR027608) T 83 Desulfotomaculum nigrificans DSM574 (X62176) Thermincola carboxydiphila 2204T (AY603000) Cryptanaerobacter phenolicus DSM15808T (AY327251) 99 Heliobacterium chlorum DSM 3682T (M11212) 73 99 Heliobacillus mobilis 6T (AB100835) Clostridia, Clostridiales, Heliorestis daurensis BT-H1T (AF079102) T Peptococcaceae/Heliobacteriaceae 86 99 Desulfitobacterium hafniense DSM 10664 (NR074996) Desulfitobacterium chlororespirans DSM 11544T (U68528) 99 Desulfosporosinus orientis DSM 765T (Y11570) 99 Desulfosporosinus auripigmenti DSM 13351T (AJ493051) Uncultured bacterium clone b19-147 (JX576081)

99 Uncultured clone OPB54 (AF027087) Hydrogenispora ethanolica LX-BT (AB669474) 52 Class/ Order OPB54 99 Uncultured bacterium clone HAW-R60-B-745d-BD (FN436105) 81 Uncultured bacterium clone AKAU3574 (DQ125579) 99 Uncultured bacterium clone PW185 (GQ402678) T 99 DSM 11254 (U82327) Moorella humiferrea DSM 23265T (GQ872425) Clostridia, Thermoanaerobacterales 1 T 52 Syntrophothermus lipocalidus TGB-C1 (AB021305) T Clostridia, Clostridiales, 99 Syntrophomonas sapovorans OM (AF022249) 99 Syntrophomonas wolfei subsp. wolfei DSM 2245T (NR074750) Syntrophomonadaceae Thermolithobacter ferrireducens JW/KA-2T (AF282254) 99 Thermolithobacter carboxydivorans DSM 7242T (DQ095862) Thermolithobacteria 96 Carboxydothermus ferrireducens DSM 11255T (U76363) 99 Carboxydothermus hydrogenoformans Z-2901T (NR074395) 64 Carboxydothermus pertinax Ug1T (AB573433) Thermacetogenium phaeum PBT (AB573433) T 99 Thermovorax subterraneus 70B (EU370564) 99 Caldanaerovirga acetigignens DSM 18802T (EF530069) 99 Thermovenabulum ferriorganovorum Z-9801T (AY033493) Tepidanaerobacter syntrophicus JLT (AB106353) T 51 99 KC4 (NR075034) Clostridia, Thermoanaerobacterales 2 Ammonifex thiophilus DSM 19636T (EF554597) Thermoanaeromonas toyohensis ToBET (AB062280) 55 T 95 Caldanaerobacter subterraneus 2707 (EF554599) 99 T 74 Caldanaerobacter subterraneus subsp. tengcongensis MB4 (NR074701) Caldanaerobacter uzonensis K67T (NR115985) 98 Thermoanaerobacter italicus Ab9T (AJ250846) T 96 Thermoanaerobacter wiegelii DSM 10319 (X92513) 99 Thermoanaerobacter sulfurophilus DSM 11584T (Y16940) Hydrogenibacillus schlegelii DSM 2000T (Z26934) Calditerricola yamamurae YMO722T (AB308475) T 51 Caldalkalibacillus thermarum HA6 (AY753654) Bacilli 2 T 81 Thermoactinomyces vulgaris DSM 43016 (AF138739) 99 Planifilum fulgidum 500275T (AB088362) 80 Thermaerobacter subterraneus C21T (AF343566) 69 Thermaerobacter litoralis KW1T (AY936496) 62 Thermaerobacter nagasakiensis DSM14512T (AB061441) 99 Thermaerobacter marianensis DSM12885T (AB011495) Thermaerobacter composti Ni80T (AB454087) Sulfobacillus acidophilus DSM 10332T (NR074758) T 99 Sulfobacillus thermosulfidooxidans BC1 (U75648) 99 Sulfobacillus sibiricus N1T (NR042730) 63 Symbiobacterium ostreiconchae KY38T (AB361629) 69 Symbiobacterium thermophilum IAM 14863T (NR075044) 82 'Symbiobacterium toebii' SC-1T (AF190460) 99 Symbiobacterium turbinis KY46T (AB455238) 93 Symbiobacterium terraclitae KA13T (AB455239) Caldinitratiruptor microaerophilus CA62NT (GQ405534) 78 Uncultured bacterium, clone SMQ31 (AM930328) 55 Uncultured bacterium, clone SMG130 (AM930312) T 52 Limnochorda pilosa strain HC45 (AB992259) 99 Uncultured compost bacterium, clone FS1689 (FN667168) Limnochordia 77 98 Uncultured bacterium, clone SMQ8 (AM93322) 99 Uncultured bacterium, clone SMG3 (AM930272) classis. nov. Uncultured compost bacterium, clone FS1639 (FN667155) Rubrobacter xylanophilus DSM 9941T (NR074552) T 77 Gaiella occulta F2-233 (JF423906) T 90 Streptomyces albosporeus NBRC 15386 (AB184637) T 99 Streptomyces exfoliatus NBRC 13475 (AB184868) T 99 Leptospirillum ferriphilum DSM 14647 (AF356829) Nitrospira moscoviensis DSM 10035T (X82558) T 56 Desulfobacca acetatoxidans DSM11109 (AF002671) T Other phyla 85 Escherichia coli ATCC 11775 (X80725) T 62 Geobacter metallireducens DSM 7210 (L07834) Thermotoga naphthophila RKU-10T (NR_074952) Chloroflexus aurantiacus J-10-fl (D38365) 95 ‘Thermus kawarayensis’ KW11T (NR112160)

0.02 T 79 Virgibacillus pantothenticus B0018 (D16275) Supplementary Figure S5: 52 Bacillus subtilis DSM 10T (AJ276351) 99 Pullulanibacillus naganoensis DSM 10191T (AB021193) ME tree based on 16S rRNA T Bacilli 1 86 Lysinibacillus boronitolerans DSM 17140 (AB199591) 62 Marinibacillus marinus 581T (AJ237708) gene sequences 56 Brevibacillus brevis ATCC 8249T (AB271756) Erysipelothrix inopinata MF-EP02T (AJ550617) Erysipelotrichia 99 Erysipelothrix rhusiopathiae ATCC 19414T (AB034200) Selenomonas sputigena ATCC 33510T (D89882) T 99 Thermosinus carboxydivorans DSM 14886 (NR117169) Negativicutes 59 Sporomusa sphaeroides DSM 2875T (AJ279801) 74 Desulfotomaculum acetoxidans 5575T (NR027608) T 81 Desulfotomaculum nigrificans DSM574 (X62176) Thermincola carboxydiphila 2204T (AY603000) Cryptanaerobacter phenolicus DSM15808T (AY327251) 99 Heliobacterium chlorum DSM 3682T (M11212) 70 99 Heliobacillus mobilis 6T (AB100835) Clostridia, Clostridiales, Heliorestis daurensis BT-H1T (AF079102) T Peptococcaceae/Heliobacteriaceae 85 99 Desulfitobacterium hafniense DSM 10664 (NR074996) Desulfitobacterium chlororespirans DSM 11544T (U68528) 99 Desulfosporosinus orientis DSM 765T (Y11570) 99 Desulfosporosinus auripigmenti DSM 13351T (AJ493051) Uncultured bacterium clone b19-147 (JX576081)

99 Uncultured clone OPB54 (AF027087) T 51 Hydrogenispora ethanolica LX-B (AB669474) Class/ Order OPB54 99 Uncultured bacterium clone HAW-R60-B-745d-BD (FN436105) 80 Uncultured bacterium clone AKAU3574 (DQ125579) 99 Uncultured bacterium clone PW185 (GQ402678) T 99 Moorella glycerini DSM 11254 (U82327) Moorella humiferrea DSM 23265T (GQ872425) Clostridia, Thermoanaerobacterales 1 T 51 Syntrophothermus lipocalidus TGB-C1 (AB021305) T Clostridia, Clostridiales, 99 Syntrophomonas sapovorans OM (AF022249) 99 Syntrophomonas wolfei subsp. wolfei DSM 2245T (NR074750) Syntrophomonadaceae Thermolithobacter ferrireducens JW/KA-2T (AF282254) 99 Thermolithobacter carboxydivorans DSM 7242T (DQ095862) Thermolithobacteria 97 Carboxydothermus ferrireducens DSM 11255T (U76363) 99 Carboxydothermus hydrogenoformans Z-2901T (NR074395) 63 Carboxydothermus pertinax Ug1T (AB573433) Thermacetogenium phaeum PBT (AB573433) 99 Thermovorax subterraneus 70BT (EU370564) 99 Caldanaerovirga acetigignens DSM 18802T (EF530069) 99 Thermovenabulum ferriorganovorum Z-9801T (AY033493) Tepidanaerobacter syntrophicus JLT (AB106353) Ammonifex degensii KC4T (NR075034) 52 99 Clostridia, Thermoanaerobacterales 2 Ammonifex thiophilus DSM 19636T (EF554597) Thermoanaeromonas toyohensis ToBET (AB062280) 54 T 93 Caldanaerobacter subterraneus 2707 (EF554599) 99 T 74 Caldanaerobacter subterraneus subsp. tengcongensis MB4 (NR074701) Caldanaerobacter uzonensis K67T (NR115985) T 98 Thermoanaerobacter italicus Ab9 (AJ250846) T 96 Thermoanaerobacter wiegelii DSM 10319 (X92513) T 99 Thermoanaerobacter sulfurophilus DSM 11584 (Y16940) Hydrogenibacillus schlegelii DSM 2000T (Z26934) Calditerricola yamamurae YMO722T (AB308475) Caldalkalibacillus thermarum HA6T (AY753654) Bacilli 2 T 79 Thermoactinomyces vulgaris DSM 43016 (AF138739) 99 Planifilum fulgidum 500275T (AB088362) T 81 Thermaerobacter subterraneus C21 (AF343566) 68 Thermaerobacter litoralis KW1T (AY936496) 61 Thermaerobacter nagasakiensis DSM14512T (AB061441) 99 Thermaerobacter marianensis DSM12885T (AB011495) Thermaerobacter composti Ni80T (AB454087) Sulfobacillus acidophilus DSM 10332T (NR074758) T 99 Sulfobacillus thermosulfidooxidans BC1 (U75648) T 99 Sulfobacillus sibiricus N1 (NR042730) T 63 Symbiobacterium ostreiconchae KY38 (AB361629) 69 Symbiobacterium thermophilum IAM 14863T (NR075044) 82 'Symbiobacterium toebii' SC-1T (AF190460) 99 Symbiobacterium turbinis KY46T (AB455238) 93 Symbiobacterium terraclitae KA13T (AB455239) Caldinitratiruptor microaerophilus CA62NT (GQ405534) 77 Uncultured bacterium, clone SMG130 (AM930312) 54 Uncultured bacterium, clone SMQ31 (AM930328) T 51 Limnochorda pilosa strain HC45 (AB992259) 99 Uncultured compost bacterium, clone FS1689 (FN667168) Limnochordia 78 97 Uncultured bacterium, clone SMQ8 (AM93322) 99 Uncultured bacterium, clone SMG3 (AM930272) classis. nov. Uncultured compost bacterium, clone FS1639 (FN667155) Rubrobacter xylanophilus DSM 9941T (NR074552)

76 Gaiella occulta F2-233 T (JF423906) T 92 Streptomyces albosporeus NBRC 15386 (AB184637) 99 Streptomyces exfoliatus NBRC 13475T (AB184868) T 99 Leptospirillum ferriphilum DSM 14647 (AF356829) Nitrospira moscoviensis DSM 10035T (X82558) T 56 Desulfobacca acetatoxidans DSM11109 (AF002671) T Other phyla 86 Escherichia coli ATCC 11775 (X80725) T 62 Geobacter metallireducens DSM 7210 (L07834) Thermotoga naphthophila RKU-10T (NR_074952) Chloroflexus aurantiacus J-10-fl (D38365) 96 ‘Thermus kawarayensis’ KW11T (NR112160)

0.02 Supplementary Table S1.

Cellular fatty acid content (percentage of total) of strain HC45T

Fatty acids Percent

C 3-OH 0.22 10:0 iso-C 3-OH 2.76 11:0 C 2-OH 0.40 11:0 C12:0 2-OH 0.26 iso-C13:0 2.40 iso-C13:0 3-OH 2.85 anteiso-C13:0 2.84 iso-C14:0 3-OH 0.71 iso-C14:0 4.57 iso-C15:0 30.99 iso-C15:0 3-OH 2.84 anteiso-C15:0 31.65

C15:0 2-OH 1.77 iso-C16:0 2.92

C16:0 4.66 iso-C17:0 2.47 anteiso-C17:0 2.17

C17:0 1.14

C18:1 ω 9c 0.54

C18:0 0.62

Summed Feature 3 1.22

Summed Feature 3 : C16:1 ω 7c / C16:1 ω 6c