1
2
3
Fig. S1 Rarefaction 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
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Table S1 Formulation of fermentation media
Medium
Ingredients (per litre)
1
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), NH 4SO4 (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), ZnSO 4·7H2O (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)
20
21
22
23
24
25
26
27
28
29
Table S2 Order level composition of actinobacterial communities from eight NL sediments. Values
represent number of sequences assigned to each order. Values in brackets represent percentage of
actinomycete community composed of each family
2
Sediment Sample
Order within
Actinobacteria
(class)
Acidimicrobiales
Actinomycetales
Coriobacteriales
Euzebyales
Solirubrobacterales
Unclassified
1L
2L
3L
4L
5L
6 (29)
9 (43)
16 (46)
4 (11)
12 (31)
13 (33)
81 (50)
57 (35)
8 (42)
3 (15)
5 (3)
1 (5)
5 (24)
15 (43)
14 (36)
18 (11)
8 (42)
6L
7L
8L
54 (51)
9 (9)
1 (1)
2 (2)
8 (42)
3 (15.8)
14 (4)
322 (91)
1 (5)
7 (37)
8 (2)
10 (2)
40 (38)
30
31
32
33
34
Table S3 Family 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 each family
Sediment Sample
Family within
Actinomycetales
1L
2L
3L
4L
5L
6L
7L
8L
Cellulomonadaceae
5
(2)
Demequinaceae
15
(5)
Geodermatophilaceae
1 (11)
146
(45)
Kineosporiaceae
Microbacteriaceae
1 (11)
Mycobacteriaceae
5 (56)
7 (12)
1
(33)
Nocardioidaceae
15
(26)
Propionibacteriaceae
1
(2)
Unclassified
Actinomycetales
3 (33)
TOTAL
9
(100)
1
(0.3)
1 (11)
4
(100)
4
(100)
13
(100)
13
(100)
34
(60)
57
(100)
3
(100)
3
(100)
7 (78)
9
(100)
2
(67)
3
(100)
155
(48)
322
(100)
35
36
37
Table S4 Membership of multi-member OTUs identified from eight NL sediments on the basis of ≥99%
sequence identity
3
OTU
No. of
strains
Fig. 2
Clade
Isolates in OTU
1
70
E3
2
3
2
3
D2
E2
4
13
C
5
6
7
8
9
6
8
2
6
4
D1
E3
E3
E2
D2
10
20
D2
11
10
E3
12
14
D2
13
8
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
1LA4, 1LD3
1LA5, 2LB7, 8LD1
1LA8, 1LC2, 2LC2, 2LC8, 4LC2, 5LA2, 5LB8, 6LA3,6LA7, 6LA10, 6LC3, 6LD5,
8LD4
1LB5, 2LMC2, 6LMC5 6LB5, 6LC10, 8LD5
1LB8, 2LA7, 2LA10, 2LB9, 2LD10, 3LB10, 4LA9, 4LC9
1LB9, 8LB7
1LB12, 1LC7, 2LB1, 8LA6, 8LC8, MISCC3
1LB4, 1LD4, 6LC5, 8LC2
2LA3, 2LA9, 2LB10, 2LC7, 2LD6, 2LD7, 2LD11, 6LC1, 7LA5,7LA7, 7LB1,7LB3,
7LB9, 7LC5, 7LC6, 7LC7, 7LC9, 7LD1, 7LD4, 7LD5
1LC10, 2LA1, 2LA5, 3LA3, 4LC1, 5LA6, 6LB1,6LC6, 6LD9, 8LD2
1LA3, 1LC1, 2LA6, 2LA8, 3LMA12, 3LD12, 4LD6 5LB1,5LB4, 5LB5, 6LA4, 8LA2,
8LB10, 8LC1
2LB4, 2LB5, 2LB6, 3LB11, 4LB2, 4LC4, 5LA9, 7LC2
14
12
E3
1LB7, 2LA11,2LB12, 3LA6, 3LB2, 4LA3, 6LA9, 6LC2, 6LC8, 6LE3, 7LC4, 8LC7
15
16
3
2
E3
E3
17
54
D2
3LB7, 4LA10, 6LC12
4LA1, 5LA3
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
19
20
21
22
23
24
25
3
6
4
10
5
9
5
E1
C
E2
D2
E3
E2
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
5LA1, 6LA6, 6LC4
1LMA10, 6LB3, 8LA1, 8LA5, 8LB3, 8LB4
2LB2, 3LA1, 6LB4, 7LA8
2LD5, 5LA12, 7LB7, 7LB5, 7LB8, 7LC1, 7LC10, 7LC11, 7LD2, 7LD11
2LC11, 7LB4, 7LC3, 7LD6, 7LD7
1LB1, 2LA2, 2LC1, 2LD1, 6LB11, 6LE4, 7LA1, 7LD9, 8LD6
1LC3, 1LC4, 1LC6, 2LC4, 2LD4
4
38
39
Table S5 Actinomycete 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. 2
clade
Seq.
length
(bp)
1
70
E3
818
2
2
D2
405
3
3
E2
451
4
13
C
470
5
6
D1
446
6
8
E3
461
7
2
E3
448
8
6
E2
446
9
4
D2
399
10
20
D2
461
11
10
E3
455
12
14
D2
462
13
8
E3
456
14
12
E3
452
15
3
E3
502
16
2
E3
398
17
54
D2
511
18
47
E3
502
19
3
E1
448
20
6
C
21
4
22
Closest GenBank Match
(Accession number)
%
ID
(OTU representative)
Streptomyces griseus strain ABRIINW
13 (HQ597007.1)
99
KM102189 (4LA5)
Streptomyces sp. HB116 (GU213500.1)
99
KM102190 (1LD2)
99
KM102191 (8LD1)
99
KM102192 (6LA10)
99
KM102193 (1LB5)
99
KM102194 (2LA10)
99
KM102195 (8LB7)
99
KM102196 (2LB1)
99
KM102197 (6LC5)
100
KM102198 (7LC9)
99
KM102199 (3LA3)
99
KM102200 (2LA8)
99
KM102201 (5LA9)
100
KM102202 (3LB2)
99
KM102203 (6LC12)
Streptomyces drozdowiczii PhyCEm1349 (AM921646.1)
Nocardiopsis sp. TFS72-17
(HM001279.1)
Streptomyces sp. CNQ-105_SD01
(EU214928.1)
Streptomyces longispororuber strain
NRRL B-3736T (DQ442521.1)
Streptomyces sp. SXY83 (GU045536.1)
Streptomyces sp. DSM 21069
(FJ872524.1)
Streptomyces albidoflavus strain HD103 (EF620360.1)
Streptomyces albidoflavus strain HD109 (EF620361.1)
Streptomyces bottropensis strain
cfcc3173 (GQ258687.1)
Streptomyces champavatii strain NRRL
B-5682 (DQ026642.1)
Streptomyces sp. AKB-2008-ET11
strain AKB-2008-ET11 (AM988883.1)
Streptomyces sp. LYG-1 (JF727260.1)
Accession Number
Streptomyces sp. AKB-2008-ET11
strain AKB (AM988883.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces albidoflavus strain HD109 (EF620361.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces peucetius strain JCM 9920
(NR_024763.1)
99
KM102204 (5LA3)
100
KM102205 (8LC3)
100
KM102206 (3LD2)
457
Nocardiopsis sp. TFS 90 (EF209052.1)
99
KM102208 (8LB4)
E2
487
Streptomyces sp. 219840 (HQ992732.1)
100
KM102209 (2LB2)
10
D2
459
Streptomyces albidoflavus strain HD109 (EF620361.1)
99
KM102210 (7LC11)
23
5
E3
458
Streptomyces sp. HB132 (GQ863908.1)
99
KM102211 (2LC11)
24
9
E2
450
98
KM102212 (1LB1)
25
5
E3
457
99
KM102213 (2LD4)
Streptomyces phaeochromogenes strain
NRRL B-2031 (EU594476.1)
Streptomyces parvus strain moss18,
isolate 18 (FR846235.1)
5
99
KM102207 (5LA1)
OTU
No. of
Strains
Fig. 2
clade
Seq.
length
(bp)
1LB10
1
E3
449
1LC9
1
E3
316
1LD1
1
D2
359
2LA4
1
E3
2LA12
1
2LB3
Closest GenBank Match
(Accession number)
%
ID
(OTU representative)
99
KM102214
96
KM102215
Streptomyces sp. 030603 (FJ842612.1)
99
KM102216
462
Streptomyces baarnensis strain NRRL
B-1902 (EF178688.1)
98
KM102218
D2
437
Streptomyces sp. Nimi (EU139307.1)
99
KM102217
1
E3
392
98
KM102220
2LB11
1
C
449
99
KM102219
2LD2
1
D2
372
98
KM102221
2LD8
1
C
453
99
KM102222
2LD9
1
E3
458
99
KM102223
3LA7
1
E3
439
99
KM102224
3LB12
1
E3
310
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
Streptomyces flavogriseus
(FR832623.1)
100
KM102228
7LA2
1
D2
402
Streptomyces sp. 030603 (FJ842612.1)
99
KM102230
7LA3
1
E3
439
99
KM102231
7LA9
1
E3
381
99
KM102232
7LA12
1
E3
446
99
KM102229
7LB2
1
E3
459
99
KM102234
7LB6
1
E3
519
100
KM111605
7LB10
1
E3
427
98
KM102233
7LD8
1
E3
273
99
KM102236
7LD10
1
E3
425
98
KM102235
8LA7
1
E2
446
99
KM102238
8LA12
1
E2
445
100
KM102237
Streptomyces globosus strain 12620-1
(EF371433.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces piomogenus strain O27
16S (GU166433.1)
Nocardiopsis sp. TFS72-17
(HM001279.1)
Streptomyces drozdowiczii strain OSAC01 (FN649464.1)
Nocardiopsis sp. TFS72-17
(HM001279.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces flavogriseus
(FR832623.1)
Streptomyces piomogenus strain O27
(GU166433.1)
Streptomyces flavogriseus ATCC 33331
(CP002475.1)
Streptomyces sp. HB132 (GQ863908.1)
Streptomyces sp. AKB-2008-ET11
(AM988883.1)
Streptomyces griseus strain ABRIINW
13 (HQ597007.1)
Streptomyces sp. SirexAA-E
(CP002993.1)
Streptomyces sp. YIM G-Q-14
(AY299621.1)
Streptomyces griseus subsp. griseus
NBRC 13350 DNA (AP009493.1)
Streptomyces beijiangensis isolate 5-41, clone psychro1 (FR837628.1)
Streptomyces zaomyceticus strain 14125
(EF063456.1)
6
Accession Number
OTU
No. of
Strains
Fig. 2
clade
Seq.
length
(bp)
8LB5
1
D2
316
8LB6
1
E3
8LC4
1
8LC5
Closest GenBank Match
(Accession number)
%
ID
(OTU representative)
Streptomyces violascens strain S11A-1
(HQ238294.1)
98
KM102239
440
Streptomyces sp. RX17 (GQ342585.1)
99
KM102240
E3
425
Streptomyces sp. Nm5 (AJ308577.1)
99
KM102242
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
99
KM102241
1LMC
1
1
A
359
99
KM102245
2L868
1
B
426
99
KM102246
Streptomyces flavogriseus
(FR832623.1)
Promicromonospora sp. ME02-6983B
(EU080946.1)
Actinoalloteichus hymeniacidonis strain
HPA177 (DQ144222.1)
Accession Number
40
41
42
Table S6. Cytotoxic metabolites identified from Actinoalloteichus sp. 2L868. Activities at a test
concentration of 20 g mL-1 are shown
Compound/Fraction (retention time)
HTB26
BJ
Saturated 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
43
44
45
7