Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters among taxonomically close Streptomyces strains.

To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T, while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.

Introduction

A large number of bioactive secondary metabolites have been found from actinomycetes[1,2]. In past years, each secondary metabolite producer was taxonomically identified at the species level based on morphological, cultural, physiological and chemical features. Consequently, correlation data between each species and its secondary metabolites are steadily being accumulated. For example, Streptomyces griseus, Streptomyces avermitilis and Streptomyces tsukubensis are well known to produce streptomycin, avermectin and tacrolimus, respectively[3-5]. However, taxonomic position of producing strains of new secondary metabolites are usually determined at the genus level based on their 16S rRNA gene sequences, while species-level assignment is not always done in the field of natural product research. Although species-level classification of secondary metabolite producers gives crucial information for researchers who are seeking new microbial compounds, relationship between species names and secondary metabolites is unclear for most cases.

Genome analyses of actinomycetes are revealing that various biosynthetic gene clusters (BGCs) for secondary metabolites are encoded in their genomes and about half to three quarters of the clusters are associated with nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways[6], which suggests that nonribosomal peptides, polyketides and their hybrid compounds are the major secondary metabolites of actinomycetes. These compounds often show pharmaceutically useful bioactivities, and many have been developed into various drugs such as antibiotics, anticancer agents, and immunosuppressants. Hence, recently, genome analysis focused on NRPS and PKS gene clusters is often employed to evaluate actinomycete strains for their ability of secondary metabolite production[7-10].

A marine-derived Streptomyces sp. TP-A0882 produces butyrolactol[11]. We recently identified the gene clusters responsible for butyrolactol and thiazostatin biosynthesis in this strain using whole genome analysis[12]. In the present study, we sequenced the genomes of three type strains taxonomically closely related to strain TP-A0882, and conducted in silico DNA-DNA hybridization (DDH) to identify this strain at the species level. We further analyzed secondary metabolite-BGCs (smBGCs) such as NRPS and PKS gene clusters in each of the genomes to elucidate the diversity of secondary metabolite-biosynthetic pathways among the taxonomically close species and provide information useful for researchers screening Streptomyces strains for new compounds.

Results

Taxonomic identification of butyrolactol-producing Streptomyces sp. TP-A0882

The 16S rRNA sequence of Streptomyces sp. TP-A0882 showed >99% nucleotide similarity to those of S. diastaticus subsp. ardesiacus NRRL B-1773T (99.9%, 1464/1465), S. coelicoflavus NBRC 15399T (99.4%, 1455/1464), and S. rubrogriseus LMG 20318T (99.0%, 1448/1462). Next, we sequenced the genomes of S. diastaticus subsp. ardesiacus NBRC 15402T, S. coelicoflavus NBRC 15399T, and S. rubrogriseus NBRC 15455T and compared them with the previously sequenced genome of Streptomyces sp. TP-A0882 to estimate their DNA-DNA relatedness values. As shown in Table 1, the DDH estimate for the comparison between Streptomyces sp. TP-A0882 and the S. diastaticus subsp. ardesiacus type strain was 94.4%. Because the probability that the DDH estimate value exceeds 70% was calculated as 97.1% (Table 1), these two strains were confirmed to belong to the same species. On the other hand, the DDH estimates between Streptomyces sp. TP-A0882 and the other taxonomically close species were lower than 46%. Therefore, we identified Streptomyces sp. TP-A0882 as S. diastaticus subsp. ardesiacus.

Table 1

Genome sequencing and digital DNA-DNA hybridization (DDH) values estimated by GGDC 2.1.

Strain	Reads (Mb)	No. of scaffolds	Genome size (bp)	G+C content (%)	Accession no.	GLM-based DDH estimate (Probability that the value exceeds 70%)b
						1	2	3	4
Streptomyces sp. TP-A0882 (NBRC 110030)a (1)	723.0	34	8,106,535	72.5	BBOK01000000	—	94.4% (97.1%)	45.1% (8.4%)	43.2% (5.8%)
S. diastaticus subsp. ardesiacus NBRC 15402T (2)	1005.0	32	7,851,547	72.7	BEWC01000000		—	45.4% (8.8%)	43.2% (5.7%)
S. coelicoflavus NBRC 15399T (3)	645.8	41	8,727,276	72.2	BEWB01000000			—	45.7% (9.5%)
S. rubrogriseus NBRC 15455T (4)	896.2	21	8,454,317	72.2	BEWD01000000				—

aData from our previous study[12].

bDistances are inferred using Formula 2 (identities/high-scoring segment pair (HSP) length) from the set of HSPs representing the most unique matches obtained by comparing each pair of genomes. These distances are transformed into values analogous to the DDH using a generalized linear model inferred from an empirical reference dataset comprising real DDH values and genome sequences.

NRPS and PKS gene clusters

In our previous study, we sequenced the genome of Streptomyces sp. TP-A0882 and identified BGCs for butyrolactol and thiazostatin[12]. The genome contains at least 14 gene clusters coding for proteins involved in NRPS and PKS pathways (Table 2). To validate whether taxonomically close strains share similar secondary metabolite biosynthetic pathways, in the current study we surveyed the NRPS and PKS gene clusters in the genomes of S. diastaticus subsp. ardesiacus NBRC 15402T, S. coelicoflavus NBRC 15399T, and S. rubrogriseus NBRC 15455T.

Table 2

Open reading frames (ORFs) encoding nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) in NRPS and PKS gene clusters from Streptomyces sp. TP-A0882 (NBRC 110030).

Gene cluster	Presumed product	ORF (accession)a	Size (aa)	Domain organization
nrps-1	coelibactin	12–265 (WP_055468803)c	554	A(dhb)
		12–266 (WP_055468804)	2,246	T-C/A/T-C/A(cys)/T
		12–267 (WP_055468805)	1,857	C/A(cys)/MT/T-TE
nrps-2	coelichelin	12–104 (WP_055468733)	3,644	A(orn)/T/E-C/A(thr)/T/E-C/A(orn)/T
nrps-3	mCys-Val-…-x-Ser	13–1 (in BBOK01000009) b	>2,354	C/A(cys)/MT/T-C/A(val)…
		22–1 (in BBOK01000019) b	>2,560	…E-C/A/T-C/A(ser)/T
nrps-4	thiazostatin	2–333 (WP_055468178)	1,829	C/A(cys)/MT/T-TE
		2–328 (WP_055468176)	1,523	T-C/A(cys)/T
		2–326 (WP_053639878)	532	A(dhb)
pks/nrps-1	x-Val-Pro-pk	10–54 (WP_055469571)	1,303	C/A/T-TE
		10–53 (WP_063788334)	3,113	A(val)/T-C/A(pro)/T-KS/KR/ACP-TE
t1pks-1	butyrolactol	10–11 (WP_055469545)c	6,065	AT/ACP-KS/AT(mm)/DH/ER/KR/ACP-KS/AT(m)/KR/ACP-KS/AT(m)/DH/KR/ACP
		10–14 (WP_055469666)	2,083	KS/AT(m)/DH/ER/KR/ACP
		10–15 (WP_055469548)	3,365	KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP
		10–16 (WP_055469549)	3,462	KS/AT/DH/ER/KR/ACP-KS/KR/ACP
		10–17 (WP_055469550)	3,135	KS/AT(m)/KR/ACP-KS/AT(m)/KR/ACP
		10–18 (WP_055469551)	1,169	KS/DH/KR/ACP
t1pks-2	AHBA-diketide	2–307 (WP_055468168)	2,191	CoL(AHBA)/KR/ACP-KS/AT(m)/ACP
		2–306 (WP_055468167)	1,296	KS/AT(m)/ACP-TE
t1pks-3	unknown	18–62 (WP_063788240)	128	ACP
		18–61 (WP_055468074)	2,027	KS/AT(m)/DH/ER/KR/ACP
		18–60 (WP_051849763)	482	KS
other t1pks(s)	unknown(s)	26–1 (WP_055470054)	>1,045	…AT(m)/DH/KR/ACP
		26–2 (WP_055470053)	1,715	KS/AT(mm)/KR/ACP
		26–3 (in BBOK01000023) b	>2,325	KS/AT(m)/DH/KR/ACP-KS…
		13–248 (WP_055468920)	>354	…DH/KR/ACP
		13–247 (WP_055468919)	3,111	KS/AT(m)/DH/KR/ACP-KS/AT(m)/ACP-TE
		13–232 (WP_055468914)	5,409	ACP-KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP
		13–231 (WP_055468913)	1,862	KS/AT(m)/DH/KR/ACP
t2pks-1	gray spore pigment	7–97 (WP_030402764)	423	KS
		7–98 (WP_053637533)	422	KS
		7–99 (WP_030402766)	89	ACP
t2pks-2	kinamycin-like	15–178 (WP_031082067)	423	KS
		15–179 (WP_031184969)	407	KS
		15–180 (WP_030402549)	89	ACP
t3pks-1	THN	4–414 (WP_031081839)	374	KS
t3pks-2	phenolic lipid	7–128 (WP_037824347)	390	KS
t3pks-3	unknown	4–314 (WP_030398736)	361	KS

Abbreviations: A, adenylation; ACP, acyl carrier protein; AHBA, aminohydroxybenzoic acid; AMT, aminotransferase; AT, acyltransferase; C, condensation; CoL, CoA ligase; DH, dehydratase; E, epimerization; ER, enoylreductase; F, formyltransferase; KR, ketoreductase; KS, ketosynthase; m, malonyl-CoA: mCys, methyl-cysteine; mGly, methyl-glycine; mm, methylmalonyl-CoA; MT, methyltransferase; pk, moiety derived from PKS pathway; T, thiolation; TD, termination; TE, thioesterase; THN, tetrahydroxynaphthalene; x, unidentified amino-acid; y, unknown building block because A domain is not present in the module. Predicted substrates of A, AT, and CoL domains are shown in brackets.

aORFs are shown as a combination of scaffold number and ORF number. Incompletely sequenced ORFs are shown in italics, and undetermined domains are shown as “…”.

bBecause the ORFs are not registered in GenBank, accession numbers for the DNA sequences encoding each ORF are instead indicated in brackets.

cEncoded on the complementary strand.

S. diastaticus subsp. ardesiacus NBRC 15402T harbors four NRPS gene (nrps) clusters, one hybrid PKS/NRPS gene (pks/nrps) cluster, at least four type I PKS gene (t1pks) clusters, two type II PKS gene (t2pks) clusters, and three type III PKS gene (t3pks) clusters, as shown in Tables 3 and 4. The number and types of gene clusters are same as those of Streptomyces sp. TP-A0882 and the sequences show >99% amino acid sequence identity to those of Streptomyces sp. TP-A0882 (NBRC 110030) based on BLAST analysis in all cases except ORF77-1 and ORF80-1 (Table 4). The structures of predicted products of the gene clusters from NBRC 15402T also matched those of TP-A0882. These results suggested that the two S. diastaticus subsp. ardesiacus strains contain identical NRPS and PKS pathways.

Table 3

Numbers of secondary metabolite-biosynthetic gene clusters (smBGCs) encoded in each genome.

smBGC for	S. diastaticus subsp. ardesiacus		S. coelicoflavus NBRC 15399T	S. rubrogriseus NBRC 15455T
	TP-A0882	NBRC 15402T
nonribosomal peptide (NRP)	4	4	4	4
hybrid polyketide (PK)/NRP	1	1	2	1
PK, type-I	>4a	>4	—b	>3
PK, type-II	2	2	3	2
PK, type-III	3	3	1	2
subtotal	>14	>14	10	>12
bacteriocin	1	1	1	1
butyrolactone	—	—	1	1
ectoine	1	1	1	1
indole	1	1	1	1
lantipeptide	1	—	3	2
melanin	1	1	1	1
oligosaccharide	1	1	—	—
siderophore, non-NRP	2	2	2	3
terpene	6	6	6	5
others	—	—	2	—
subtotal	14	13	18	15
total	>28	27	28	27

aAs some type-I PKS gene clusters were not completely sequenced, exact numbers are unclear.

bNot detected.

Table 4

ORFs encoding NRPSs and PKSs in NRPS and PKS gene clusters from S. diastaticus subsp. ardesiacus NBRC 15402T.

Gene cluster	Presumed product	ORFa	Size (aa)	Domain organization	Closest homolog (accession, origin, % of identity/similarity)b
nrps-1	coelibactin	1–1240c	542	A(dhb)	WP_055468803, Streptomyces sp. NBRC 110030, 97/97
		1–1241	2,213	T-C/A/T-C/A(cys)/T	KOX46963, Streptomyces sp. NRRL F-7442, 99/99(WP_055468804, Streptomyces sp. NBRC 110030, 99/99)
		1–1242	1,857	C/A(cys)/MT/T-TE	WP_053663986, Streptomyces sp. NRRL F-7442, 99/99(WP_055468805, Streptomyces sp. NBRC 110030, 99/99)
nrps-2	coelichelin	1–1084	3,644	A(orn)/T/E-C/A(thr)/T/E-C/A(orn)/T	KOX26695, Streptomyces sp. NRRL F-4707, 99/99(WP_055468733, Streptomyces sp. NBRC 110030, 99/99)
nrps-3	mCys-Val-x-x-Ser	1–84	3,616	C/A(cys)/MT/T-C/A(val)/T/E-C	WP_051908973, Streptomyces sp. NRRL F-5635, 99/99d
		1–85	3,241	A/T/E-C/A/T-C/A(ser)/T	EHN79578, Streptomyces coelicoflavus ZG0656, 93/94d
nrps-4	thiazostatin	10–229	1,829	C/A(cys)/MT/T-TE	WP_031081050, Streptomyces sp. NRRL S-1831, 99/99(WP_055468178, Streptomyces sp. NBRC 110030, 99/99)
		10–234	1,535	T-C/A(cys)/T	WP_031184402, Streptomyces sp. NRRL F-5635, 99/98(WP_055468176, Streptomyces sp. NBRC 110030, 98/98)
		10–236	532	A(dhb)	WP_053639878, Streptomyces sp. NBRC 110030, 99/99
pks/nrps-1	x-Val-Pro-pk	5–41	1,303	C/A/T-TE	WP_055469571, Streptomyces sp. NBRC 110030, 99/99
		5–40	3,105	A(val)/T-C/A(pro)/T-KS/KR/ACP-TE	WP_063788334, Streptomyces sp. NBRC 110030, 99/99
t1pks-1	butyrolactol	43–30c	6,062	AT/ACP-KS/AT(mm)/DH/ER/KR/ACP-KS/AT(m)/KR/ACP-KS/AT(m)/DH/KR/ACP	WP_055469545, Streptomyces sp. NBRC 110030, 99/99
		43–33	>398	KS…	WP_055469666, Streptomyces sp. NBRC 110030, 98/98
		58–1	>1,655	…AT/DH/ER/KR/ACP	WP_055469666, Streptomyces sp. NBRC 110030, 98/98
		58–2	>426	KS…	WP_055469548, Streptomyces sp. NBRC 110030, 99/99
		62–1	>1,601	…AT(m)/DH/KR/ACP-KS…	WP_055469548, Streptomyces sp. NBRC 110030, 97/98
		5–1	>1,334	…AT(m)/DH/KR/ACP	WP_055469548, Streptomyces sp. NBRC 110030, 97/98
		5-2	3,464	KS/AT/DH/ER/KR/ACP-KS/KR/ACP	WP_055469549, Streptomyces sp. NBRC 110030, 99/99
		5-3	3,141	KS/AT(m)/DH/KR/ACP-KS/AT(m)/KR/ACP	KOT98773, Streptomyces sp. NRRL F-4711, 99/99(WP_055469550, Streptomyces sp. NBRC 110030, 99/99)
		5-4	1,169	KS/DH/KR/ACP	KOT98774, Streptomyces sp. NRRL F-4711, 99/99(WP_055469551, Streptomyces sp. NBRC 110030, 99/99)
t1pks-2	AHBA-diketide	10–255	2,191	CoL(AHBA)/KR/ACP-KS/AT(m)/ACP	WP_051908920, Streptomyces sp. NRRL F-5635, 99/99(WP_055468168, Streptomyces sp. NBRC 110030, 99/99)
		10–256	1,296	KS/AT(m)/ACP-TE	WP_053663292, Streptomyces sp. NRRL F-7442, 99/99(WP_055468167, Streptomyces sp. NBRC 110030, 99/99)
t1pks-3	unknown	6–621	128	ACP	WP_063788240, Streptomyces sp. NBRC 110030, 99/100
		6–622	2,027	KS/AT(m)/DH/ER/KR/ACP	KOX28560, Streptomyces sp. NRRL F-4707, 99/99(WP_055468074, Streptomyces sp. NBRC 110030, 99/100)
		6–623	482	KS	WP_051783751, Streptomyces sp. NRRL F-5555, 99/99(WP_051849763, Streptomyces sp. NBRC 110030, 99/99)
other t1pks(s)	unknown(s)	53-3	>1,507	…AT(m)/DH/KR/ACP	WP_055470054, Streptomyces sp. NBRC 110030, 99/98
		53-2	1,650	KS/AT(mm)/KR/ACP	KOX41189, Streptomyces sp. NRRL F-7442, 99/99(WP_055470053, Streptomyces sp. NBRC 110030, 99/99)
		53-1	>2,463	KS/AT(m)/DH/KR/ACP-KS/AT…	WP_033305239, Streptomyces atroolivaceus, 57/67d
		77-1	>762	…KR/ACP-KS…	AHH99923, Kutzneria albida DSM 43870, 63/74
		80-1	>489	…KS…	WP_040741646, Nocardia tenerifensis, 70/78
		59-1	>350	…KR/ACP	WP_055468920, Streptomyces sp. NBRC 110030, 100/100
		59-2	>1,370	KS/AT(m)/DH…	WP_055468919, Streptomyces sp. NBRC 110030, 99/99
		20-1	>1,605	…KR/ACP-KS/AT(m)/ACP-TE	WP_055468919, Streptomyces sp. NBRC 110030, 99/99
		20-16	5,412	ACP-KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP	WP_053639270, Streptomyces sp. NRRL F-4707, 99/99(WP_055468914, Streptomyces sp. NBRC 110030, 99/99)
		20-17	1,862	KS/AT(m)/DH/KR/ACP	WP_053639271, Streptomyces sp. NRRL F-4707, 99/99(WP_055468913, Streptomyces sp. NBRC 110030, 99/99)
t2pks-1	gray spore pigment	2–814	423	KS	WP_030402764, Streptomyces sp. NBRC 110030, 100/100
		2–813	422	KS	WP_053637533, Streptomyces sp. NBRC 110030, 99/100
		2–812	89	ACP	WP_030402766, Streptomyces sp. NBRC 110030, 100/100
t2pks-2	kinamycin-like	15–126	423	KS	KOX34713, Streptomyces sp. NRRL F-4707, 100/100(WP_031082067, Streptomyces sp. NBRC 110030, 99/100)
		15–127	407	KS	WP_031184969, Streptomyces sp. NRRL F-5635, 99/100(WP_055468989, Streptomyces sp. NBRC 110030, 99/99)
		15–128	89	ACP	WP_030402549, Streptomyces sp. NBRC 110030, 99/100
t3pks-1	THN	8–149	374	KS	WP_031081839, Streptomyces sp. NBRC 110030, 100/100
t3pks-2	phenolic lipid	1–740	390	KS	WP_037824347, Streptomyces sp. NBRC 110030, 99/99
t3pks-3	unknown	10–248	361	KS	WP_030398736, Streptomyces sp. NBRC 110030, 100/100

Abbreviations are the same as those of Table 2.

aORFs are shown as a combination of scaffold number and ORF number. Incompletely sequenced ORFs are shown in italics, and undetermined domains are shown as “…”.

bParentheses indicate that the closest homolog is not from Streptomyces sp. TP-A0882 (NBRC 110030).

cEncoded on the complementary strand.

dAlthough homologs in Streptomyces sp. TP-A0882 did not appear as high score hits in basic local alignment search tool analyses because they are not registered in GenBank, they are present in scaffolds 13 (BBOK01000009), 22 (BBOK01000019), and 26 (BBOK01000023) of the Streptomyces sp. TP-A0882 genome.

S. coelicoflavus NBRC 15399T harbors four nrps clusters, two pks/nrps clusters, three t2pks clusters, and one t3pks cluster, as shown in Table 5. Unlike typical Streptomyces strains, t1pks cluster is not present in this strain. nrps-i, nrps-ii, pks/nrps-i, t2pks-i, and t3pks-i were predicted to be responsible for the synthesis of coelibactin, coelichelin, prodiginine, gray spore pigment, and tetrahydroxynaphthalene (THN), respectively, based on high similarities (85–99% amino acid sequence identity) to SCO7681-7683, SCO0492 (CchH), SCO5886-SCO5894 (Red), SCO5318-SCO5316 (WhiE), and SCO1206 (RppA) of Streptomyces coelicolor A3(2)[6,13], respectively. Based on the domain and module organizations and substrate selective residues in the A domains, nrps-iii and nrps-iv were predicted to synthesize nonribosomal peptides consisting of eight amino acids and 13 amino acids, respectively. The product of pks/nrps-ii was speculated to be a novel oxazolomycin analog because the domain organization is similar, but not identical, to that of the BGCs for oxazolomycins[14]. Although the remaining two gene clusters (t2pks-ii, t2pks-iii) are likely to be responsible for the synthesis of aromatic polyketides, the structures were not predicted from the sequence information alone. Analysis of the genome sequence of S. coelicoflavus strain ZG0656, the only S. coelicoflavus strain of which genome sequence is published[15], indicated that all of the S. coelicoflavus NBRC 15399T gene clusters (Table 5) are present also in strain ZG0656 with >97% amino acid sequence identity based on BLAST comparisons.

Table 5

ORFs encoding NRPSs and PKSs in NRPS and PKS gene clusters of S. coelicoflavus NBRC 15399T.

Gene cluster	Presumed product	ORFa	Size (aa)	Domain organization	Closest homolog (accession, origin, % of identity/similarity)b
nrps-i	coelibactin	3–140c	554	A(dhb)	EHN75391, S. coelicoflavus ZG0656, 99/99(CAC17498, S. coelicolor A3(2), 85/89)
		3–141	2,250	T-C/A/T-C/A(cys)/T	EHN75408, S. coelicoflavus ZG0656, 99/99(CAC17499, S. coelicolor A3(2), 86/89)
		3–142	1,857	C/A(cys)/MT/T-TE	EHN75409, S. coelicoflavus ZG0656, 99/99(CAC17500, S. coelicolor A3(2), 89/91)
nrps-i	coelichelin	6–362	3,666	A(orn)/T/E-C/A(thr)/T/E-C/A(orn)/T	KPC76200, Streptomyces sp. NRRL WC-3753, 99/99(EHN78004, S. coelicoflavus ZG0656, 99/98;CAB53322, S. coelicolor A3(2), 86/90)
nrps-iii	x-x-Ser-mCys-Val-x-x-Ser	3–549	3,637	C/A(cys)/MT/T-C/A(val)/T/E-C/A/T/E-C/A/T-C/A(ser)/T	EHN75118, S. coelicoflavus ZG0656, 99/99
		3–550	3,271	A/T/E-C/A/T-C/A(ser)/T	EHN79578, S. coelicoflavus ZG0656, 99/98
nrps-iv	Gly-y-Asp-Tyl-Thr-x-Asp-Gly-Pro-Gly-Gly-Ala-mGly	2–543	6,937	C/A(gly)/T-C/T-C/A(asp)/T/E-C/A(tyl)/T-C/A(thr)/T-C/A/T/E	KPC71694, Streptomyces sp. NRRL WC-3753, 99/99(EHN72150, S. coelicoflavus ZG0656, 99/99)
		2–544	4,213	C/A(asp)/T-C/A(gly)/T-C/A(pro)/T-C/A(gly)/T	KPC71705, Streptomyces sp. NRRL WC-3753, 99/99(EHN72136, S. coelicoflavus ZG0656, 99/99)
		2–545	3,865	C/A(gly)/T-C/A(ala)/T-C/A(gly)/MT/T-TE	WP_054100963, Streptomyces sp. NRRL WC-3753, 99/99(EHN72117, S. coelicoflavus ZG0656, 99/99)
pks/nrps-i	prodiginine	3–99	1,012	KS/KS	KPC87173, Streptomyces sp. NRRL WC-3753, 99/99(EHN75481, S. coelicoflavus ZG0656, 99/99;CAA16487, S. coelicolor A3(2), 91/94)
		3–107c	407	KS	EHN75487, S. coelicoflavus ZG0656, 100/100(CAA16177, S. coelicolor A3(2), 94/97)
		3–108c	81	ACP	EHN75488, S. coelicoflavus ZG0656, 100/100(CAA16178, S. coelicolor A3(2), 96/97)
		3–110	87	ACP	EHN77254, S. coelicoflavus ZG0656, 100/100(CAA16180, S. coelicolor A3(2), 95/95)
		3–111	636	ACP/AMT	KPC87185, Streptomyces sp. NRRL WC-3753, 99/98(EHN75478, S. coelicoflavus ZG0656, 98/98;CAA16181, S. coelicolor A3(2), 88/89)
		3–112	532	A(cys)	KPC87186, Streptomyces sp. NRRL WC-3753, 99/99(EHN75480, S. coelicoflavus ZG0656, 99/99;CAA16182, S. coelicolor A3(2), 93/95)
		3–113	2,306	CoL(NH2)/T-KS/AT(m)/ACP/AMT	EHN77210, S. coelicoflavus ZG0656, 99/99(CAA16183, S. coelicolor A3(2), 87/90)
		3–115	280	TE	EHN75475, S. coelicoflavus ZG0656, 99/99(CAA16185, S. coelicolor A3(2), 96/96)
pks/nrps-ii	oxazolomycin-like	7–245c	842	ACP-TD	WP_051005867, S. coelicoflavus ZG0656, 98/98
		7–244c	1,752	KS/ACP-C/FkbH	WP_054101954, Streptomyces sp. NRRL WC-3753, 99/98(WP_051005868, S. coelicoflavus ZG0656, 98/98)
		7–242	2,968	DH/ACP/ACP/ACP/DH-KS/KR/ACP-KS/ACP	WP_054101951, Streptomyces sp. NRRL WC-3753, 97/97(EHN75054, S. coelicoflavus ZG0656, 97/97)
		7–241	3,008	C/A(ser)/T-C/A/MT/T-C	KPC72343, Streptomyces sp. NRRL WC-3753, 99/99(EHN75030, S. coelicoflavus ZG0656, 99/99)
		7–237	4,903	KS/DH/KR/ACP-KS/DH/KR/ACP-KS/DH/KR/MT/ACP	KPC72421, Streptomyces sp. NRRL WC-3753, 98/98(EHN75036, S. coelicoflavus ZG0656, 97/97)
		7–236	1,158	F/A(gly)/T	KPC71002, Streptomyces sp. NRRL WC-3753, 99/99(EHN77489, S. coelicoflavus ZG0656, 99/99)
		7–234	879	KS/ACP	KPC71004, Streptomyces sp. NRRL WC-3753, 99/99(EHN75023, S. coelicoflavus ZG0656, 99/99)
		7–233	6,079	KS/KR/MT/ACP-C/A(gly)/T-KS/DH/KR/ACP-KS/KR/ACP-KS	WP_054102642, Streptomyces sp. NRRL WC-3753, 98/98(EHN78704, S. coelicoflavus ZG0656, 98/98)
		7–232	1,106	AT/AT(m)	EHN78700, S. coelicoflavus ZG0656, 99/99
t2pks-i	gray spore pigment	11–215	423	KS	KPC88984, Streptomyces sp. NRRL WC-3753, 100/100(EHN75824, S. coelicoflavus ZG0656, 99/99;CAB45606, S. coelicolor A3(2), 98/99)
		11–214	424	KS	KPC88985, Streptomyces sp. NRRL WC-3753, 99/99(EHN75823, S. coelicoflavus ZG0656, 98/98;CAB45607, S. coelicolor A3(2), 98/98)
		11–213	89	ACP	EHN75822, S. coelicoflavus ZG0656, 100/100(CAB45608, S. coelicolor A3(2), 98/98)
t2pks-ii	unknown	1–30	84	ACP	EHN79053, S. coelicoflavus ZG0656, 100/100
		1–31	422	KS	EHN79055, S. coelicoflavus ZG0656, 100/100
		1–32	416	KS	EHN79056, S. coelicoflavus ZG0656, 99/99
t2pks-iii	unknown	14–63	421	KS	EHN77732, S. coelicoflavus ZG0656, 100/100
		14–62	415	KS	KPC71304, Streptomyces sp. NRRL WC-3753, 99/99(EHN77731, S. coelicoflavus ZG0656, 99/99)
t3pks-i	THN	5–164	374	KS	EHN79529, S. coelicoflavus ZG0656, 100/100(CAC01488, S. coelicolor A3(2), 91/95)

Abbreviations are the same as those of Table 2.

aORFs are shown as a combination of scaffold number and ORF number.

bIf the homolog in S. coelicoflavus ZG0656 is not the closest and/or Streptomyces coelicolor A3(2) harbors the homolog, it is shown in parentheses.

cEncoded on the complementary strand.

S. rubrogriseus NBRC 15455T harbors four nrps clusters, one pks/nrps cluster, at least three t1pks clusters, two t2pks clusters, and two t3pks clusters (Table 6). nrps-a, nrps-b, nrps-c, pks/nrps-a, t1pks-a, t1pks-b, t2pks-a, t3pks-a, and t3pks-b were predicted to be responsible for the synthesis of coelibactin, coelichelin, calcium-dependent antibiotic (CDA), prodiginine, coelimycin, eicosapentaenoic acid, gray spore pigment, THN, and phenolic acid, respectively, based on high similarities (91–100% amino acid sequence identities) to SCO7681-7683, SCO0492 (CchH), SCO3230-SCO3032 (CDA peptide synthetases), SCO5886-SCO5894 (Red), SCO6275-SCO6273 (Cpk), SCO0126-SCO0127, SCO5318-SCO5316 (WhiE), SCO1206 (RppA), and SCO7671 (SrsA ortholog)[6,13], respectively. Based on the domain and module organization and substrate selective residues in the A domains, nrps-d was predicted to synthesize a peptide containing cysteine. Other t1pks cluster(s) were not completely sequenced, but their predicted PKS proteins do not have high sequence similarity to the known PKS proteins, suggesting that the product(s) might be novel. t2pks-b is likely to synthesize aromatic polyketides, but the products could not be predicted because the sequence does not show a high degree of similarity to any PKS whose products have been elucidated. Among the 12 gene clusters, all except the other t1pks genes and t2pks-b show >93% sequence similarity to the corresponding genes from S. coelicolor A3(2), suggesting that most of the gene clusters in S. rubrogriseus NBRC 15455T are present also in S. coelicolor A3(2).

Table 6

ORFs encoding NRPSs and PKSs in NRPS and PKS gene clusters of S. rubrogriseus NBRC 15455T.

Gene cluster	Presumed product	ORFa	Size (aa)	Domain organization	Closest homolog (accession, origin, % of identity/similarity)b
nrps-a	coelibactin	7–361c	553	A(dhb)	CAC17498, S. coelicolor A3(2), 98/98
		7–362	2,240	T-C/A/T-C/A(cys)/T	CAC17499, S. coelicolor A3(2), 96/97
		7–363	1,842	C/A(cys)/MT/T-TE	CAC17500, S. coelicolor A3(2), 97/97
nrps-b	coelichelin	4–1115	3,649	A(orn)/T/E-C/A(thr)/T/E-C/A(orn)/T	CAB53322, S. coelicolor A3(2), 95/96
nrps-c	CDA	13–202	7,395	C/A(ser)/T-C/A(thr)/T-C/A(trp)/T/E-C/A(asp)/T-C/A(asp)/T-C/A(hpg)/T/E	CAB38518, S. coelicolor A3(2), 95/96
		13–203	3,658	C/A(asp)/T-C/A(gly)/T-C/A(asn)/T/E	CAB38517, S. coelicolor A3(2), 96/97
		13–204	2,429	C/A/T-C/A(trp)/T-TE	CAD55498, S. coelicolor A3(2), 97/97
nrps-d	x-y-Cys	1–88	1,177	A/T-C/T	SDT78734, Streptomyces sp. 2114.2, 98/98(CAA18918, S. coelicolor A3(2), 98/98)
		1–89	1,413	C/A(cys)/T-TE	CAA18919, S. coelicolor A3(2), 96/96
pks/nrps-a	prodiginine	3–389	932	KS/KS	CAA16487, S. coelicolor A3(2), 96/96
		3–381c	407	KS	CAA16177, S. coelicolor A3(2), 99/99
		3–380c	81	ACP	CAA16178, S. coelicolor A3(2), 99/100
		3–378	87	ACP	CAA16180, S. coelicolor A3(2), 100/100
		3–377	641	ACP/ACP/AMT	CAA16181, S. coelicolor A3(2), 97/97
		3–376	532	A(cys)	CAA16182, S. coelicolor A3(2), 98/99
		3–375	2,298	CoL/T-KS/AT(m)/ACP/AMT	SDT77027, Streptomyces sp. 2114.2, 96/96(CAA16183, S. coelicolor A3(2), 95/96)
		3–373	280	TE	CAA16185, S. coelicolor A3(2), 98/100
t1pks-a	coelimycin	1–2	4,563	KS/AT(m)/ACP-KS/AT(m)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP-	SDT78409, Streptomyces sp. 2114.2, 93/95(CAD55506, S. coelicolor A3(2), 98/98)
		1-1	>595	KS…	CAC22145, S. coelicolor A3(2), 94/97
		34-1	>1,743	…AT/DH/KR/ACP-KS…	CAC22145, S. coelicolor A3(2), 92/95
		36-1	>907	…DH/KR/ACP	CAC22145, S. coelicolor A3(2), 91/93
		36-2	>582	KS…	CAC22144, S. coelicolor A3(2), 96/97
t1pks-b	EPA	7–477	2,074	KS/AT(m)/ACP/KR/DH	SDS27436, Streptomyces sp. 2114.2, 96/96(CAB52353, S. coelicolor A3(2), 95/95)
		7–476	2,240	KS/AT	CAB52354, S. coelicolor A3(2), 96/97
other t1pks(s)	unknown(s)	6-1	>1,561	KS/AT/ACP-KS…	SCE45938, Streptomyces sp. DvalAA-14, 74/80
		35-1	>933	…DH/KR/ACP	APD71595, Streptomyces sp. MM3, 54/65
		35-2	1,622	KS/AT(mm)/KR/ACP	AJC56296, Streptomyces sp. 769, 52/64
		35-3	>493	KS…	APD71977, Streptomyces sp. MM3, 69/81
		9–576	>1,388	…AT(mm)/KR/ACP-TE	SCD97877, Streptomyces sp. DvalAA-14, 66/75
		9–571c	693	KS/ACP	WP_052397599, Streptomyces sp. NRRL F-5123, 75/81
		9–569c	3,992	KS/ACP-KS/AT(mm)/DH/KR/ACP-KS/AT(m)/DH/KR/ACP	CDR05500, Streptomyces iranensis, 48/58
t2pks-a	gray spore pigment	10–372	423	KS	CAB45606, S. coelicolor A3(2), 98/99
		10–371	424	KS	CAB45607, S. coelicolor A3(2), 99/99
		10–370	90	ACP	CAB45608, S. coelicolor A3(2), 100/100
t2pks-b	unknown	9–560	82	ACP	WP_031518191, Streptomyces sp. NRRL F-5123, 81/89
		9–561	421	KS	WP_031518190, Streptomyces sp. NRRL F-5123, 85/91
		9–562	421	KS	WP_033177057, Streptomyces sp. URHA0041, 86/91
t3pks-a	THN	4–335	374	KS	SDS82518, Streptomyces sp. 2114.2, 96/98(CAC01488, S. coelicolor A3(2), 96/98)
t3pks-b	phenolic acid	7–351	391	KS	CAC17488, S. coelicolor A3(2), 90/91

CDA, calcium-dependent antibiotic; EPA, eicosapentaenoic acid. The other abbreviations are the same as those of Table 2.

aORFs are shown as a combination of scaffold number and ORF number. Incompletely sequenced ORFs are shown in italics, and undetermined domains are shown as “…”.

bParentheses indicate that the closest homolog is not from S. coelicolor A3(2).

cEncoded on the complementary strand.

Conservation of NRPS and PKS gene clusters among taxonomically close species

As summarized in Fig. 1a, BGCs for coelibactin, coelichelin, gray spore pigment, and THN are present in all of the strains. The prodiginine biosynthetic gene (red) cluster is not present in S. diastaticus subsp. ardesiacus strains NBRC 15402T and TP-A0882, but is present in both S. coelicoflavus NBRC 15399T and S. rubrogriseus NBRC 15455T. The phenolic lipid biosynthetic gene (srs) cluster is present in both S. diastaticus subsp. ardesiacus strains and S. rubrogriseus NBRC 15455T. Products of the nrps-3 cluster from the S. diastaticus subsp. ardesiacus strains and the nrps-iii cluster from S. coelicoflavus NBRC 15399T include mCys-Val-x-x-Ser. However, their products are actually not the same (S. diastaticus subsp. ardesiacus strains, mCys-Val-x-x-Ser; S. coelicoflavus NBRC 15399T, x-x-Ser-mCys-Val-x-x-Ser). Overall, the S. diastaticus subsp. ardesiacus strains, S. coelicoflavus NBRC 15399T, and S. rubrogriseus NBRC 15455T harbor at least eight, four, and six species-specific gene clusters, respectively.

Figure 1

Schematic diagram showing diversity of NRPS & PKS gene clusters (a) and the other biosynthetic gene clusters (b) in the taxonomically close species. As nrps-3 of the S. diastaticus subsp. ardesiacus strains and nrps-iii of S. coelicoflavus NBRC 15399T show partial sequence similarity, the diagram shows putative sharing between these two species. However, the gene products of nrps-3 and nrps-iii are divergent (mCys-Val-x-x-Ser and x-x-Ser-mCys-Val-x-x-Ser, respectively). Abbreviations: CDA, calcium-dependent antibiotic; EPA, eicosapentaenoic acid; GPS, gray spore pigment; m, methyl-; NIS, NRPS-independent siderophore; pk, moiety derived from PKS pathway; THN, tetrahydroxynaphthalene; x, unidentified amino-acid; y, unknown building block. aThe lantipeptide BGC, whose precursors peptide sequences are AVLINLDhbDDGCGDhaDhbCDhaDhaPCADhbNVA and CNGDhaCADhbNVA, is not present in the genome of of S. diastaticus subsp. ardesiacus NBRC 15402T; bincluding desferrioxamine; calbaflavenone, hopene, carotenoid & gosmin.

The other secondary metabolite-biosynthetic gene clusters

In addition to NRPS and PKS gene clusters, the other smBGCs were also investigated. Thirteen to 18 gene clusters are encoded in each genome as shown in Table 3. Table 7 lists the clusters with putative products and loci. Homologous gene clusters are aligned in the same row in the table. S. diastaticus subsp. ardesiacus TP-A0882 and NBRC 15402T shared the same set of gene clusters, except for a BGC for lantipeptides, suggesting that the two strains contain almost identical secondary metabolite biosynthetic pathways. Among the 18 BGCs of S. coelicoflavus NBRC 15399T, 13 are present also in S. coelicoflavus strain ZG0656 whereas three lantipeptide and two terpene BGCs are not. All 15 BGCs identified from S. rubrogriseus NBRC 15455T are present also in S. coelicolor A3(2) (data not shown). BGCs for bacteriocin, ectoine, indole melanine, two siderophores, four terpenes are sheared among the three species, whereby 3 to 5 BGCs are specific in each species (Table 7, Fig. 1b).

Table 7

Loci encoding the other smBGCs in the draft genome sequences.

smBGC for	Putative product (Most similar known cluster)a	S. diastaticus subsp. ardesiacus		S. coelicoflavus NBRC 15399T	S. rubrogriseus NBRC 15455T
		TP-A0882	NBRC 15402T
Bacteriocin	Informatipeptin	3,881–14,096, s07b	1,033,543–1,043,758, s01	109,209–119,424, s06	825,412–835,627, s04
Butyrolactone	unidentifiedc	NDd	ND	143,762–189,159, s14	ND
Butyrolactone	—e	ND	ND	ND	1–8,053, s03
Ectoine	Ectoine	299,195–309,593, s13	68,465–78,863, s18	229,038–239,436, s12	666,768–677,166, s06
Indole	unidentified	53,333–74,460, s16	737,576–758,703, s10	123,011–144,138, s13	127,502–148,620, s07
Lantipeptide	2 or 3 kinds of peptidesf	337,776–362,051, s07	ND	33,290–58,341, s23	ND
Lantipeptide	GLVNLDhbDDNCGDhaDhbCGACDhbDhbNVAg	ND	ND	143,762–189,159, s14	ND
Lantipeptide	unidentified	ND	ND	582,852–607,263, s03	ND
Lantipeptide	DhbGDhaRADhaLLLCGDDhaDhaLDhaIDhbDhbCNg	ND	ND	ND	400,232–422,952, s01
Lantipeptide	AQFGEGDhbFDhbDhaPDhaDhaYAIGDhbRCPICCg	ND	ND	ND	1,370,541–1,405,635, s04
Melanin	Melanin	343,594–354,160,s02	476,879–487,445, s09	291,936–302,595, s01_2	359,033–369,602, s09
Oligosaccharide	unidentified	1–24,831, s10	225,676–260,720, s15	ND	ND
Siderophore	Desferrioxamine B	258,147–269,916, s02	561,212–572,981, s09	191,379–203,157, s01_2	266,567–278,345, s09
Siderophore	—	138,812–150,764, s15	57,784–69,736, s15	26,019–38,040, s03	549,038–560,963, s03
Siderophore	unidentified	ND	ND	ND	11,549–66,510, s03
Terpene	Albaflavenone	200,318–221,331,s01	778,230–799,243, s02	137,667–158,680, s11	301,202–322,287, s10
Terpene	Hopene	408,409–435,138, s11	560,033–586,762, s01	21,764–48,513, s07	495,633–522,374, s01
Terpene	Carotenoid	119,566–143,614, s16	668,873–692,929, s10	44,866–68,934, s13	44,374–68,462, s07
Terpene	Geosminh	160,585–182,786, s09	190,986–213,187, s20	424,806–447,016, s03	207,587–229,767, s03
Terpene	unidentified	17,748–38,641, s19	161,086–181,979, s01	ND	ND
Terpene	—	241,318–265,214, s09	201,308–225,195, s05	ND	ND
Terpene	2–methylisoborneol	ND	ND	306,224–319,060, s13	ND
Terpene	Isorenieratene	ND	ND	110,932–136,512, s20	ND
Terpene	—	ND	ND	ND	1–20,497, s22
Other	Lomaiviticin	ND	ND	100,475–140,891, s14	ND
Other	unidentified	ND	ND	18,943–60,076, s18	ND

aWhen the outputs of antiSMASH showed >40% gene similarities, we putatively considered them as putative products; bLocus is shown as start-end positions and scaffold no. (sxx means scaffold000xx); cAs analysis using antiSMASH output product names but the gene similarities were less 40% gene similarity, the products are shown as unidentified; dNot detected; eNo output; fAVLINLDhb(didehydrobutyrine)DDGCGDha(didehydroalanine)DhbCDhaDhaPCADhbNVA & CNGDhaCADhbNVA in S. diastaticus subsp. ardesiacus TP-A0882, DhaDGGCGDhaDhbCGNACIDhaDhaGDha, INLDhbDDGCGDhaDhbCDhaDhaPCADhbNVA & CKGDhaCADhbNVA in S. coelicoflavus NBRC 15399T; gCore peptide amino acid sequence predicted by antiSMASH; hbased on the similarity to BGCs for giosmin.

Discussion

Genome analysis conducted in this study shows that S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T share an almost identical set of smBGCs, while S. coelicoflavus strains NBRC 15399T and ZG0656 shared their own similar set of gene clusters. Previous studies on Nocardia brasiliensis[8] and Salinispora species[16] have also shown that most smBGCs are common within each species, with strain-specific ones being relatively limited. These results suggest that actinomycete strains belonging to the same species are also likely to possess similar secondary metabolite biosynthetic pathways.

In contrast, only a limited number of smBGC are shared by different species examined in this study, even though they have >99% 16S rRNA gene sequence similarity and are thus considered taxonomically close. We identified totally 49 different smBGCs including 25 NRPS and PKS gene clusters from the three species. Among them, 14 clusters, responsible for production of coelibactin, coelichelin, gray spore pigment, THN, bacteriocin, ectoine, indole, melanin, two types of NRPS-independent siderophres, and four types of terpenes are conserved among the three species, while additional five clusters for phenolic lipid, prodiginine, nonribosomal peptide, lantipeptide, and terpene syntheses are shared by two species. Coelibactin and coelichelin are iron-chelating molecules, known as siderophores, that are involved in uptake of ferric iron[17]. Like gray spore pigment and melanin, THN is involved in pigmentation, as it is used in melanin formation[18]. Pigment production is often examined in taxonomic studies[19]. Phenolic lipids are components of the cell wall, and are involved in resistance to β-lactam antibiotics by affecting the characteristics and rigidity of the cytoplasmic membrane/peptidoglycan[20]. Ectoine is an osmolyte and involved in protection against extreme osmotic stress[21]. Therefore, many of the conserved/shared gene clusters identified in this study are physiologically and/or taxonomically important. The remaining 33 smBGCs are species-specific, with each of the three species containing different eleven specific clusters.

Unexpectedly, most of the gene clusters in S. rubrogriseus NBRC 15455T are present also in S. coelicolor (correctly classified as Streptomyces violaceoruber)[22] A3(2). As the sequence similarities in these regions are very high (>93%), we considered it possible that strains NBRC 15455T and A3(2) might actually be the same species. To clarify this, we conducted in silico DDH analysis of the two genome sequences. The resulting estimated DDH value is 70.3% (67.3–73.2%), which is just on the borderline between two strains belonging to the same or different species, and the probability that the value exceeds 70% was calculated to be 78.9% (data not shown). Orthologs of the other t1pks cluster(s) and t2pks-b found in S. rubrogriseus NBRC 15455T (Table 6) were not identified in S. coelicolor A3(2), while orthologs of SCO5073-SCO5092 (actinorhodin), SCO6826-SCO6827, SCO7669-SCO7671 (aromatic polyketide), SCO7221 (germicidin), SCP1.228c-SCP1.246 (methylenomycin), SCO0381-SCO0401, and SCO7700-SCO7701 (2-methylisoborneol) present in S. coelicolor A3(2), could not be identified in S. rubrogriseus NBRC 15455T. These findings indicated that strains NBRC 15455T and A3(2) are likely to be separate species. Very recently, phylogenetic relationships among Streptomyces species were examined using multi-locus sequence analysis. The study showed that S. violaceoruber was distinct from S. rubrogriseus[23], supporting our current conclusion.

Here, we have shown an example that actinomycetes strains belonging to the same species share a conserved set of smBGCs, whereas different species each harbor species-specific smBGCs in addition to some common ones even if the species are taxonomically close. Relationships between species and smBGCs in actinomycetes were reported by Doroghazi et al.[24], Ziemert et al.[16], and Seipke et al.[25]. As the study by Doroghazi et al. is a large-scale analysis for taxonomically diverse 840 actinobacterial strains encompassing many genera, they did not compare smBGCs between taxonomically close Streptomyces species. Ziemert et al. reported the diversity and evolution of PKS and NRPS gene clusters within the genus Salinispora. In contrast to rare actinomycetes such as Salinispora, relationships between species and smBGCs are less well elucidated in the genus Streptomyces. Seipke et al. showed strain-level diversity of smBGCs in S. albus. However, the strains were actually not S. albus[23] and may not belong to a single species but be divided into two independent genomospecies whose in silico DDH value is less 70% (our unpublished data). As the genus Streptomyces includes many species, accumulation of data for more Streptomyces species is needed to clarify whether smBGCs are diverse at strain-level or conserved at species-level. As reported here, genome sequence-based analysis will provide more insight into relationships between Streptomyces species and their secondary metabolites.

Methods

Strains

Streptomyces diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T were obtained from the NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan) culture collection. Streptomyces sp. TP-A0882 has been deposited into the NBRC culture collection and registered as NBRC 110030[12].

Analysis of 16S rRNA gene sequences

The 16S rRNA genes were amplified using two universal primers, 9F and 1541R, and sequenced according to an established method[26]. EzTaxon-e was used for basic local alignment search tool (BLAST) analysis of the sequences[27].

Genome sequencing

Genomic DNA was prepared from each of the strains as described previously[28]. The prepared DNA was subjected to paired-end sequencing using the MiSeq sequencing system (Illumina, San Diego, CA, USA) as per the manufacturer’s instructions. The sequence redundancies for the three draft genomes were 74-128-fold. The sequence reads were assembled using Newbler v2.8 (454 Life Sciences, Branford, CT, USA) and subsequently finished using GenoFinisher[29].

In silico DDH

DNA-DNA relatedness values were estimated from the genome sequences using Genome-to-Genome Distance Calculator (GGDC) 2.1, available from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) website (http://ggdc.dsmz.de/distcalc2.php)[30].

Analysis of NRPS and PKS gene clusters

Coding regions in the draft genome sequences were predicted using Prodigal v2.6[31]. NRPS and PKS gene clusters were determined as previously reported[9,10]. A BLASTP search was performed using the NCBI Protein BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins), in which the non-redundant protein sequence (nr) database was chosen as the Search Set. AntiSMASH[32] was used to predict substrates for adenylation, acyltransferase, and CoA ligase domains.

Analysis of the other secondary metabolite biosynthetic gene clusters

BGCs except for PKS and NRPS gene clusters in the draft genome sequences were searched using antiSMASH[32].

Nucleotide accession numbers

The draft genome sequences in this study were deposited in GenBank/EMBL/DDBJ under the accession numbers shown in Table 1.