Fig. S1Rarefaction curves showing effect of sampling depth on the number of actinobacterial species level OTUs (D=0.03). The lines for samples 2L, 3L, 4L, 5L and 7L overlap in the graph. Error bars indicate 95% confidence intervals
Figure prepared in Microsoft Excel and exported from Excel as a .png
Table S1Formulation of fermentation media
Medium / Ingredients (per litre)BFM1 / beef extract (10 g), CaCO3 (2 g), dextrin (20 g), (NH4)2SO4 (2 g), peptone (5 g), soluble starch, Instant Ocean (18 g)
BFM2 / pharmamedia (5 g), soluble starch (5 g), Instant Ocean (18 g)
BFM3 / agar (0.4 g), glycerol (12 g), K2HPO4 (3 g), KCl (0.5 g), MgSO4·7H20 (0.5 g), NaCl (5 g), soy peptone (5 g), Instant Ocean (18 g)
BFM4 / agar (0.4 g), CaCO3 (1 g), dextrose (12 g), NH4Cl (1 g), nutrisoy (12 g), NZ-amine A (3 g), Instant Ocean (18 g)
Mn / Fe(NH4)2(SO4)2 (0.15 g), MnCO3 (1 g), Na4P2O7-10 H20 (0.05 g), sodium citrate (0.15 g), yeast extract (0.075 g), Instant Ocean (18 g)
SGC / casein-peptone (5 g), corn steep powder (10 g), glucose (10 g), soluble starch (10 g), yeast extract (2 g), Instant Ocean (18 g)
AA / casein hydrolysate (2 g), glucose (2 g), glycine (0.5 g), lysine (0.5 g), NH4SO4 (0.5 g), ornithine (0.5 g), Pharmamedia (5 g), soluble starch (5 g), Instant Ocean (18 g)
ISP4 / CaCO3 (2 g), FeSO4.7H2O (1 mg), (NH4)2(SO4)2 (2 g), K2HPO4 (2 g), MgCl2 7H20 (1 mg), MgSO4·7H2O (1 g), NaCl (1 g), soluble starch (10 g), ZnSO4·7H2O (1 mg), Instant Ocean (18 g)
SED / pharmamedia (5 g), soluble starch (5 g) and Instant Ocean (18 g ) were added to 500 mL of sediment extract. Sediment extract was prepared by boiling a known weight of sediment in 500 mL of MilliQ water for 1 h followed by filtering to remove sediment particle. Sediment extracts were only made from four NL sediments because only strains derived from these sediments were fermented in SED media. Weights of sediments used to prepare sediment extracts were 2L (57.10 g), 3L (24.83 g), 5L (21.27 g) and 6L (11.64 g).
ISP2 / Difco ISP2 premix (18 g)
Table S2Order level composition of actinobacterial communitiesactinobacterial communities from eight NL sediments. Values represent number of sequences assigned to each order. Values in brackets represent percentage of actinomycete community composed of a particulareach family
Sediment SampleOrder within Actinobacteria (class) / 1L / 2L / 3L / 4L / 5L / 6L / 7L / 8L
Acidimicrobiales / 6 (29) / 16 (46) / 12 (31) / 81 (50) / 8 (42) / 54 (51) / 8 (42) / 14 (4)
Actinomycetales / 9 (43) / 4 (11) / 13 (33) / 57 (35) / 3 (15) / 9 (9) / 3 (15.8) / 322 (91)
Coriobacteriales / 1 (1)
Euzebyales / 5 (3) / 2 (2)
Solirubrobacterales / 1 (5) / 1 (5) / 8 (2)
Unclassified / 5 (24) / 15 (43) / 14 (36) / 18 (11) / 8 (42) / 40 (38) / 7 (37) / 10 (2)
Table S3Family level composition of actinomycete communities from eight NL sediments. Values represent number of sequences assigned to each family. Values in brackets represent percentage of actinomycete community composed of a particulareach family
Sediment SampleFamily within Actinomycetales / 1L / 2L / 3L / 4L / 5L / 6L / 7L / 8L
Cellulomonadaceae / 5
(2)
Demequinaceae / 15 (5)
Geodermatophilaceae / 1 (11)
Kineosporiaceae / 146 (45)
Microbacteriaceae / 1 (11) / 7 (12) / 1 (11) / 1 (0.3)
Mycobacteriaceae / 5 (56) / 1 (33)
Nocardioidaceae / 15 (26)
Propionibacteriaceae / 1
(2)
Unclassified Actinomycetales / 3 (33) / 4 (100) / 13 (100) / 34 (60) / 3 (100) / 7 (78) / 2
(67) / 155 (48)
TOTAL / 9 (100) / 4 (100) / 13 (100) / 57 (100) / 3 (100) / 9 (100) / 3
(100) / 322 (100)
Table S4 Membership of multi-member OTUs identified from eight NL sediments on the basis of ≥99% sequence identity
OTU / No. of strains / Fig. 2 Clade / Isolates in OTU1 / 70 / E3 / 1LA1, 1LB3, 1LC8, 2LC5, 2LC9, 2LC10, 2LC12, 2LD3, 3LA2, 3LA4, 3LA5, 3LA8, 3LA9, 3LA10, 3LA11,3LA12 3LB5, 3LB6, 3LB8, 3LC4, 3LC5, 3LC6, 3LD1, 3LD4, 3LD6, 3LD7, 3LD8, 3LD9, 3LD10, 3LD11, 4LA4, 4LA5, 4LA6, 4LA8, 4LA11, 4LA12, 4LB3, 4LB5, 4LB6, 4LB7, 4LB8, 4LB9, 4LB10, 4LB11, 4LB12, 4LC7, 4LC10 , 4LC11, 4LD1, 4LD4, 4LD8, 5LA8,5LA10, 5LA11, 6LA11, 6LB7, 6LB8, 6LC7, 6LD1, 6LD10, 6LMC6, 7LA6, 7LA11, 7LB12, 7LC12, 8LA3, 8LA11, 8LB8, 8LB11, 8LMC7
2 / 2 / D2 / 1LA4, 1LD3
3 / 3 / E2 / 1LA5, 2LB7, 8LD1
4 / 13 / C / 1LA8, 1LC2, 2LC2, 2LC8, 4LC2, 5LA2, 5LB8, 6LA3,6LA7, 6LA10, 6LC3, 6LD5, 8LD4
5 / 6 / D1 / 1LB5, 2LMC2, 6LMC5 6LB5, 6LC10, 8LD5
6 / 8 / E3 / 1LB8, 2LA7, 2LA10, 2LB9, 2LD10, 3LB10, 4LA9, 4LC9
7 / 2 / E3 / 1LB9, 8LB7
8 / 6 / E2 / 1LB12, 1LC7, 2LB1, 8LA6, 8LC8, MISCC3
9 / 4 / D2 / 1LB4, 1LD4, 6LC5, 8LC2
10 / 20 / D2 / 2LA3, 2LA9, 2LB10, 2LC7, 2LD6, 2LD7, 2LD11, 6LC1, 7LA5,7LA7, 7LB1,7LB3, 7LB9, 7LC5, 7LC6, 7LC7, 7LC9, 7LD1, 7LD4, 7LD5
11 / 10 / E3 / 1LC10, 2LA1, 2LA5, 3LA3, 4LC1, 5LA6, 6LB1,6LC6, 6LD9, 8LD2
12 / 14 / D2 / 1LA3, 1LC1, 2LA6, 2LA8, 3LMA12, 3LD12, 4LD6 5LB1,5LB4, 5LB5, 6LA4, 8LA2, 8LB10, 8LC1
13 / 8 / E3 / 2LB4, 2LB5, 2LB6, 3LB11, 4LB2, 4LC4, 5LA9, 7LC2
14 / 12 / E3 / 1LB7, 2LA11,2LB12, 3LA6, 3LB2, 4LA3, 6LA9, 6LC2, 6LC8, 6LE3, 7LC4, 8LC7
15 / 3 / E3 / 3LB7, 4LA10, 6LC12
16 / 2 / E3 / 4LA1, 5LA3
17 / 54 / D2 / 1LA2, 1LA6, 1LA9, 1LA11, 1LB2, 1LB6, 1LC5, 1LC12, 1LD2, 1LD5, 2LB8, 2LC3, 2LC6, 2LD12, 3LB1, 3LB4, 3LC2,3LD5, 3LE1, 4LA2, 4LB1,4LC3, 4LC5, 4LC6,4LC12,4LD2, 4LD5, 5LA5, 5LA7, 5LB2, 5LB3, 5LB7, 6LA2, 6LB2, 6LB6, 6LB9, 6LD4, 6LD6, 6LD8,6LD12, 6LE5, 7LA4, 7LA10, 7LB11, 7LC8, 7LD3, 8LB1, 8LB2, 8LB12, 8LC3, 8LC12, 8LD3, 8LD7, MISCC4
18 / 47 / E3 / 1LA7, 1LA10, 1LA12, 1LB11, 1LC11, 2LMA11, 3LB3, 3LB9, 3LC1, 3LC3, 3LC7, 3LC8, 3LC9, 3LC10, 3LC11, 3LC12, 3LD2, 4LA7,4LB4, 4LC8,4LD3, 4LD7, 4LD9, 5LA4, 5LB6, 5LB9, 5LB10, 5LB11, 6LA5, 6LA8, 6LA12, 6LB10, 6LC9, 6LC11, 6LD2, 6LD3, 6LD7, 6LD11, 6LE1, 8LA4, 8LA8, 8LA9, 8LA10, 8LB9, 8LC6, 8LC10, 8LD8
19 / 3 / E1 / 5LA1, 6LA6, 6LC4
20 / 6 / C / 1LMA10, 6LB3, 8LA1, 8LA5, 8LB3, 8LB4
21 / 4 / E2 / 2LB2, 3LA1, 6LB4, 7LA8
22 / 10 / D2 / 2LD5, 5LA12, 7LB7, 7LB5, 7LB8, 7LC1, 7LC10, 7LC11, 7LD2, 7LD11
23 / 5 / E3 / 2LC11, 7LB4, 7LC3, 7LD6, 7LD7
24 / 9 / E2 / 1LB1, 2LA2, 2LC1, 2LD1, 6LB11, 6LE4, 7LA1, 7LD9, 8LD6
25 / 5 / E3 / 1LC3, 1LC4, 1LC6, 2LC4, 2LD4
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Table S5OTUs identified from aActinomycetes OTUs isolated from eight NL sediments on the basis of ≥99% sequence identity. Percent identity (% ID) to the closest GenBank match is shown
OTU / No. of Strains / Fig. 2clade / Seq. length(bp) / Closest GenBank Match (Accession number) / % ID / Accession Number(OTU representative)
1 / 70 / E3 / 818 / Streptomyces griseus strain ABRIINW 13 (HQ597007.1) / 99 / KM102189 (4LA5)
2 / 2 / D2 / 405 / Streptomyces sp. HB116 (GU213500.1) / 99 / KM102190 (1LD2)
3 / 3 / E2 / 451 / Streptomycesdrozdowiczii PhyCEm-1349 (AM921646.1) / 99 / KM102191 (8LD1)
4 / 13 / C / 470 / Nocardiopsis sp. TFS72-17 (HM001279.1) / 99 / KM102192 (6LA10)
5 / 6 / D1 / 446 / Streptomyces sp. CNQ-105_SD01 (EU214928.1) / 99 / KM102193 (1LB5)
6 / 8 / E3 / 461 / Streptomyceslongispororuber strain NRRL B-3736T (DQ442521.1) / 99 / KM102194 (2LA10)
7 / 2 / E3 / 448 / Streptomyces sp. SXY83 (GU045536.1) / 99 / KM102195 (8LB7)
8 / 6 / E2 / 446 / Streptomyces sp. DSM 21069 (FJ872524.1) / 99 / KM102196 (2LB1)
9 / 4 / D2 / 399 / Streptomycesalbidoflavus strain HD-103 (EF620360.1) / 99 / KM102197 (6LC5)
10 / 20 / D2 / 461 / Streptomycesalbidoflavus strain HD-109 (EF620361.1) / 100 / KM102198 (7LC9)
11 / 10 / E3 / 455 / Streptomycesbottropensis strain cfcc3173 (GQ258687.1) / 99 / KM102199 (3LA3)
12 / 14 / D2 / 462 / Streptomyceschampavatii strain NRRL B-5682 (DQ026642.1) / 99 / KM102200 (2LA8)
13 / 8 / E3 / 456 / Streptomyces sp. AKB-2008-ET11 strain AKB-2008-ET11 (AM988883.1) / 99 / KM102201 (5LA9)
14 / 12 / E3 / 452 / Streptomyces sp. LYG-1 (JF727260.1) / 100 / KM102202 (3LB2)
15 / 3 / E3 / 502 / Streptomyces sp. AKB-2008-ET11 strain AKB (AM988883.1) / 99 / KM102203 (6LC12)
16 / 2 / E3 / 398 / Streptomycesflavogriseus(FR832623.1) / 99 / KM102204 (5LA3)
17 / 54 / D2 / 511 / Streptomycesalbidoflavus strain HD-109 (EF620361.1) / 100 / KM102205 (8LC3)
18 / 47 / E3 / 502 / Streptomycesflavogriseus (FR832623.1) / 100 / KM102206 (3LD2)
19 / 3 / E1 / 448 / Streptomycespeucetius strain JCM 9920 (NR_024763.1) / 99 / KM102207 (5LA1)
20 / 6 / C / 457 / Nocardiopsis sp. TFS 90 (EF209052.1) / 99 / KM102208 (8LB4)
21 / 4 / E2 / 487 / Streptomyces sp. 219840 (HQ992732.1) / 100 / KM102209 (2LB2)
22 / 10 / D2 / 459 / Streptomycesalbidoflavus strain HD-109 (EF620361.1) / 99 / KM102210 (7LC11)
23 / 5 / E3 / 458 / Streptomyces sp. HB132 (GQ863908.1) / 99 / KM102211 (2LC11)
24 / 9 / E2 / 450 / Streptomycesphaeochromogenes strain NRRL B-2031 (EU594476.1) / 98 / KM102212 (1LB1)
25 / 5 / E3 / 457 / Streptomycesparvus strain moss18, isolate 18 (FR846235.1) / 99 / KM102213 (2LD4)
1LB10 / 1 / E3 / 449 / Streptomycesglobosus strain 12620-1 (EF371433.1) / 99 / KM102214
1LC9 / 1 / E3 / 316 / Streptomycesflavogriseus (FR832623.1) / 96 / KM102215
1LD1 / 1 / D2 / 359 / Streptomyces sp. 030603 (FJ842612.1) / 99 / KM102216
2LA4 / 1 / E3 / 462 / Streptomycesbaarnensis strain NRRL B-1902 (EF178688.1) / 98 / KM102218
2LA12 / 1 / D2 / 437 / Streptomyces sp. Nimi (EU139307.1) / 99 / KM102217
2LB3 / 1 / E3 / 392 / Streptomycespiomogenus strain O27 16S (GU166433.1) / 98 / KM102220
2LB11 / 1 / C / 449 / Nocardiopsis sp. TFS72-17 (HM001279.1) / 99 / KM102219
2LD2 / 1 / D2 / 372 / Streptomycesdrozdowicziistrain OS-AC01 (FN649464.1) / 98 / KM102221
2LD8 / 1 / C / 453 / Nocardiopsis sp. TFS72-17 (HM001279.1) / 99 / KM102222
2LD9 / 1 / E3 / 458 / Streptomycesflavogriseus (FR832623.1) / 99 / KM102223
3LA7 / 1 / E3 / 439 / Streptomycesflavogriseus (FR832623.1) / 99 / KM102224
3LB12 / 1 / E3 / 310 / Streptomycesflavogriseus (FR832623.1) / 99 / KM102225
3LD3 / 1 / E3 / 457 / Streptomyces sp. L083 (EU135902.1) / 99 / KM102226
6LB12 / 1 / D2 / 441 / Streptomyces sp. HV3 (FJ222809.1) / 99 / KM102227
6LE2 / 1 / E3 / 490 / Streptomycesflavogriseus (FR832623.1) / 100 / KM102228
7LA2 / 1 / D2 / 402 / Streptomyces sp. 030603 (FJ842612.1) / 99 / KM102230
7LA3 / 1 / E3 / 439 / Streptomycespiomogenus strain O27 (GU166433.1) / 99 / KM102231
7LA9 / 1 / E3 / 381 / Streptomycesflavogriseus ATCC 33331 (CP002475.1) / 99 / KM102232
7LA12 / 1 / E3 / 446 / Streptomyces sp. HB132 (GQ863908.1) / 99 / KM102229
7LB2 / 1 / E3 / 459 / Streptomyces sp. AKB-2008-ET11 (AM988883.1) / 99 / KM102234
7LB6 / 1 / E3 / 519 / Streptomycesgriseus strain ABRIINW 13 (HQ597007.1) / 100 / KM111605
7LB10 / 1 / E3 / 427 / Streptomyces sp. SirexAA-E(CP002993.1) / 98 / KM102233
7LD8 / 1 / E3 / 273 / Streptomyces sp. YIM G-Q-14 (AY299621.1) / 99 / KM102236
7LD10 / 1 / E3 / 425 / Streptomycesgriseus subsp. griseus NBRC 13350 DNA (AP009493.1) / 98 / KM102235
8LA7 / 1 / E2 / 446 / Streptomycesbeijiangensisisolate 5-4-1, clone psychro1 (FR837628.1) / 99 / KM102238
8LA12 / 1 / E2 / 445 / Streptomyceszaomyceticus strain 14125 (EF063456.1) / 100 / KM102237
8LB5 / 1 / D2 / 316 / Streptomycesviolascens strain S11A-1 (HQ238294.1) / 98 / KM102239
8LB6 / 1 / E3 / 440 / Streptomyces sp. RX17 (GQ342585.1) / 99 / KM102240
8LC4 / 1 / E3 / 425 / Streptomyces sp. Nm5 (AJ308577.1) / 99 / KM102242
8LC5 / 1 / D1 / 376 / Streptomyces sp. Sp080513SC-34 (AB498748.1) / 98 / KM102243
8LC9 / 1 / E2 / 447 / Streptomyces sp. BF-01 (GQ503333.1) / 98 / KM102244
8LC11 / 1 / E3 / 446 / Streptomycesflavogriseus (FR832623.1) / 99 / KM102241
1LMC1 / 1 / A / 359 / Promicromonospora sp. ME02-6983B (EU080946.1) / 99 / KM102245
2L868 / 1 / B / 426 / Actinoalloteichushymeniacidonis strain HPA177 (DQ144222.1) / 99 / KM102246
Table S6. Cytotoxic metabolites identified from Actinoalloteichus sp. 2L868. Activities at a test concentration of 20 gmL-1 are shown
Compound/Fraction (retention time) / HTB26 / BJSaturated mono-hydroxylated fatty acids (3.48 min) / 99.3 / 72.7
Saturated fatty acids (3.52 min) / 87.3 / 12.7
Saturated fatty acids (3.56 min) / 77.0 / 6.0
Polyhydroxylated unsaturated fatty acids (3.67 min) / 79.3 / 0.7
Polyhydroxylated unsaturated fatty acids (3.74 min) / 75.7 / 90.0
Glycerolipids (hydroxylated fatty acids ) (4.01 min) / 99.3 / 99.7
Glycerolipids (hydroxylated fatty acids ) (4.05 min) / 99.3 / 99.0
1-Isopentadecanoyl-3β-D-glucopyranosyl-X-Glycerol (4.00 min) / 91.0 / 2.3
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