Streptomyces lydicamycinicus sp. nov. and Its Secondary Metabolite Biosynthetic Gene Clusters for Polyketide and Nonribosomal Peptide Compounds
1
Biological Resource Center, National Institute of Technology and Evaluation (NBRC), 2-5-8 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan
2
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
*
Author to whom correspondence should be addressed.
Microorganisms 2020, 8(3), 370; https://doi.org/10.3390/microorganisms8030370
Submission received: 23 January 2020
/
Revised: 21 February 2020
/
Accepted: 2 March 2020
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Published: 6 March 2020
(This article belongs to the Special Issue Natural Products from Streptomyces)
Abstract
:(1) Background: Streptomyces sp. TP-A0598 derived from seawater produces lydicamycin and its congeners. We aimed to investigate its taxonomic status; (2) Methods: A polyphasic approach and whole genome analysis are employed; (3) Results: Strain TP-A0598 contained ll-diaminopimelic acid, glutamic acid, glycine, and alanine in its peptidoglycan. The predominant menaquinones were MK-9(H6) and MK-9(H8), and the major fatty acids were C16:0, iso-C15:0, iso-C16:0, and anteiso-C15:0. Streptomyces sp. TP-A0598 showed a 16S rDNA sequence similarity value of 99.93% (1 nucleottide difference) to Streptomyces angustmyceticus NRRL B-2347T. The digital DNA–DNA hybridisation value between Streptomyces sp. TP-A0598 and its closely related type strains was 25%–46%. Differences in phenotypic characteristics between Streptomyces sp. TP-A0598 and its phylogenetically closest relative, S. angustmyceticus NBRC 3934T, suggested strain TP-A0598 to be a novel species. Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T harboured nine and 13 biosynthetic gene clusters for polyketides and nonribosomal peptides, respectively, among which only five clusters were shared between them, whereas the others are specific for each strain; and (4) Conclusions: For strain TP-A0598, the name Streptomyces lydicamycinicus sp. nov. is proposed; the type strain is TP-A0598T (=NBRC 110027T).
1. Introduction
The genus Streptomyces is the largest taxon within the phylum Actinobacteria, and the members are an attractive source of bioactive secondary metabolites. A large number of bioactive compounds have been discovered from them, many of which have been developed into pharmaceuticals and are clinically used [1,2]. Genome analyses of Streptomyces strains revealed that each strain has a large and linear chromosome encoding more than 20 secondary metabolite biosynthetic gene clusters (smBGCs), even if it is known to produce only few secondary metabolites. This means that hitherto reported compounds are nothing more than only a part of the secondary metabolites that they can produce. One-half to three-quarters of smBGCs in Streptomyces genomes is polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters [3], suggesting polyketides and nonribosomal peptides are major secondary metabolites in this genus. Type I PKSs and NRPSs are large multifunctional enzymes with various catalytic domains, and the metabolites are synthesized according to the co-linearity rule of assembly lines. Hence, the chemical structures of the polyketide and peptide backbones can be bioinformatically predicted according to domain organizations of the gene clusters [4]. Because polyketides and nonribosomal peptides show various bioactivities, genome mining focused on PKS and NRPS gene clusters often leads to the discovery of new biologically active compounds.
In the search for new anti-methicillin-resistant Staphylococcus aureus antibiotics from marine actinomycetes, Streptomyces sp. TP-A0598 was isolated from deep sea water and found to produce lydicamycin and its new congeners, TPU-0037-A to TPU-0037-D, of polyketide origin [5]. The biosynthetic gene cluster (BGC) for these compounds was identified through analysis of its genome, and then their biosynthetic pathway was proposed [6]. In this study, we found that Streptomyces sp. TP-A0598 is phylogenetically close to Streptomyces angustmyceticus with a 16S rDNA sequence similarity of 99.93%.
Nowadays, 16S rDNA sequence analysis has conventionally been employed to identify each producer at genus-level but the producers are rarely identified at species-level in natural product studies. However, it is important to classify antibiotic producers at the species level because relationships between taxonomic species and their secondary metabolites are useful information to prioritize strains as a screening source for bioactive compounds. Thus, we investigated the taxonomic status of strain TP-A0598 using a polyphasic approach and then surveyed PKS and NRPS gene clusters in the genome. We also discuss the similarity and difference of these smBGCs among taxonomically close strains.
2. Materials and Methods
2.1. Strains
2.2. Phenotypic and Chemotaxonomic Characterization
To determine the optimal temperature and pH for growth, the strain was incubated for 7 days in BactoTM Tryptic Soy Broth (TSB; Becton, Dickinson and Company, Sparks, MD, USA) at 5 °C, 10 °C, 20 °C, 25 °C, 28 °C, 37 °C, and 45 °C and at pH 3 to pH 13, respectively. Growth in various concentrations of NaCl was also examined after 7 days of incubation in TSB. Chemotaxonomic experiments were conducted on the basis of a previous report [7]. Physiological and biochemical characteristics were evaluated using API ZYM, API Coryne, and API 50CH Biochemical Test Kits (bioMérieux, Marcy I’Etoile, France) according to the manufacturer’s instructions. Assimilation of carbon sources at a final concentration of 1% (w/v) was tested using ISP 9 agar as the basal medium according to Pridham and Gottlieb [8].
2.3. Phylogenetic Analysis Based on 16S rDNA Sequences
The 16S rDNA sequence was determined as previously described [6], and EzBioCloud was used in the similarity analysis to type strains of valid species [9]. Phylogenetic trees were constructed using the neighbour-joining [10] and the maximum likelihood methods with Kimura 2-parameter model [11] via ClustalX, MEGA X [12], and NJplot.
2.4. Genome Analysis
Genomic DNA preparation and the whole genome shotgun sequencing of S. angustmyceticus NBRC 3934T were performed as described in a previous report [6]. The sequence redundancy for the draft genome was 103-fold. The draft genome sequence was composed of 43 scaffolds with a total size of 8.09 Mb. Draft genome sequence of S. angustmyceticus NBRC 3934T has been available in GenBank/ENA/DDBJ under accession numbers BLAG01000001-BLAG01000046. Digital DNA–DNA hybridization (DDH) between the draft genome sequences of S. angustmyceticus NBRC 3934T and Streptomyces sp. TP-A0598 (BBNO01000001-BBNO01000020) was conducted using Formula 2 of Genome-to-Genome Distance Calculator [13]. Average nucleotide identity (ANI) values were calculated using the ANI Calculator (www.ezbiocloud.net/tools/ani) [14]. PKS and NRPS gene clusters were analyzed as described in a previous report [15]. Rates of PKS and NRPS gene clusters conserved between two strains (RC) were calculated as follows: RC (%) = Cab × 2 × (A + B)−1 × 100, where A, B, and Cab are the numbers of PKS and NRPS gene clusters in strain A, in strain B, and conserved between strains A and B, respectively.
3. Results
3.1. Classification of Streptomyces sp. TP-A0598
The whole-cell hydrolysate of Streptomyces sp. TP-A0598 contained ll-diaminopimelic acid, glutamic acid, glycine and alanine. The major menaquinone was MK-9(H6) and MK-9(H8), whose contents were 53% and 36%, respectively. MK-9(H2) and MK-9(H4) were also observed as minor components (each at <10%). The major cellular fatty acids (>10% of the total) were C16:0, iso-C15:0, iso-C16:0, and anteiso-C15:0. These chemotaxonomic data corresponded to the feature of the genus Streptomyces.
Streptomyces sp. TP-A0598 showed a 16S rDNA sequence similarity of 99.93% to S. angustmyceticus NRRL B-2347T (1450/1451, 1 nucleotide [nt] difference) as the closest species and formed a monophyletic clade with S. angustmyceticus in the phylogenetic trees based on 16S rDNA sequences (Figure 1). Phylogenetically close species were Streptomyces nigrescens, Streptomyces libani subsp. libani, and Streptomyces tubercidicus, which showed similarities of 99.79%, 99.79% (3 nt difference), and 99.72% (4 nt diffrenece) to strain TP-A0598, respectively. S. nigrescens and S. libani subsp. libani have been reported to be the same species [16]. These three strains of two species formed an independent clade, of which the position is close to that of TP-A0598. Streptomyces catenulae NBRC 12848T was also close to the two clades, although the bootstrap values are not high. Digital DDH indicated that DNA–DNA relatedness between Streptomyces sp. TP-A0598 and the four type strains are 25%–46%, which is much lower than the cut-off point of 70% recommended for the assignment of bacteria strains to the same genomic species [17]. ANI values between Streptomyces sp. TP-A0598 and the four type strains were 82.1–92.3%, whose values are also below the recommended threshold for species delineation (95%–96%) [18]. These results suggest Streptomyces sp. TP-A0598 to be a novel genomospecies.
Next, we characterized the strain by comparing it with its phylogenetically closest relative, S. angustmyceticus NBRC 3934T. Strain TP-A0598 formed light yellow or vivid yellow substrate mycelia and white to grey aerial mycelia and produced a vivid yellow soluble pigment when cultured on trypticase soy agar. The morphological features cultured on the other agar medium are summarized in Table 1. The pH and temperature ranges for growth were pH 5–11 and 15–37 °C, respectively. The optimum temperature was 25–28 °C. At 37 °C, abundant sporulation was observed. The strain grew in the presence of 0%–5% NaCl (w/v). Strain TP-A0598 showed morphological, chemotaxonomic, physiological, and biochemical features different from those of S. angustmyceticus NBRC 3934T (Table 2).
3.2. PKS and NRPS Gene Clusters of Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T
Streptomyces sp. TP-A0598 harbours two type I PKS (t1pks-1, t1pks-2), two type II PKS (t2pks-1, t2pks-2), one type III PKS (t3pks-1), two NRPS (nrps-1, nrps-2), and two hybrid PKS/NRPS (pks/nrps-1, pks/nrps-2) gene clusters in the genome as listed in Table 3. Pks/nrps-1 is already reported as the BGC for lydicamycins [6]. T2pks-1, t2pks-2 and t3pks-1 were identified as BGCs for spore pigment, oxytetracycline (oxy) and tetrahydroxynaphthalene (THN) (rpp), respectively. The other clusters are orphan, whose products have not been specified by previous studies. We predict the putative backbone for metabolites produced by nrps-1, nrps-2, and pks/nrps-2 as shown in Table 3 based on their module organisations and substrates of adenylation domains [4,15].
The genome of S. angustmyceticus NBRC 3934T encoded four type I PKS (t1pks-1, t1pks-2, t1pks-3, t1pks-4), two type II PKS (t2pks-1, t2pks-3), one type III PKS (t3pks-1), four NRPS (nrps-1, nrps-3, nrps-4, nrps-5) and two hybrid PKS/NRPS (nrps-3, nrps-4), as listed in Table 4. The sequences of t1pks-3 and t1pks-4 gene clusters could not be completely determined because their sequences were divided into three and seven scaffolds, respectively. However, all the open reading frames (ORFs) of t1pks-3 and t1pks-4 showed high amino acid sequence similarities to TsnB [19] (96%–98%) and ScaP [20] (89%–97%), respectively. Therefore, these gene clusters were considered as BGCs for trichostatin A and caniferolides, respectively. T2pks-1, t2pks-3, and t3pks-1 were identified as BGCs for spore pigment, trioxacarcin, and THN (rpp), respectively. Pks/nrps-3 showed similar gene organisation to that of BGC for guadinomine (gdn) [21] but did not encode all gdn genes. Hence, we predicted the product to be partial. As the other gene clusters were orphan, we predicted the backbone as shown in Table 4. Nrps-4 and nrps-5 were not be able to be completely sequenced.
Among the PKS and NRPS gene clusters found in their genomes, five (t1pks-1, t1pks-2, t2pks-1, t3pks-1, and nrps-1, highlighted in boldface in Table 3 and Table 4) were conserved between the two strains, whereas four (t2pks-2, nrps-2, pks/nrps-1, pks/nrps-2) and eight (t1pks-3, t1pks-4, t2pks-3, nrps-3, nrps-4, nrps-5, pks/nrps-3, pks/nrps-4) were specific in Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T, respectively. These numbers and the putative products are summarized in Figure 2. As Streptomyces sp. TP-A0598 harbours nine gene clusters, five of nine (56%) were the same as those of S. angustmyceticus NBRC 3934T whereas four of nine (44%) were specific to Streptomyces sp. TP-A0598. Similarly, because S. angustmyceticus NBRC 3934T possesses 13 gene clusters, five of 13 (38%) were the same as those of Streptomyces sp. TP-A0598, whereas eight of 13 (62%) are specific to S. angustmyceticus NBRC 3934T. On average, PKS and NRPS gene clusters conserved between these two strains occupy 45%. We putatively define the rate as RC (rate of PKS and NRPS gene clusters conserved between two strains) for the discussion below.
4. Discussion
The difference in 16S rDNA sequences between Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T was only 1 bp. Unexpectedly, however, the two strains were classified into different species, and Streptomyces sp. TP-A0598 was revealed to be a novel species in this study. Therefore, here, we propose the name Streptomyces lydicamycinicus sp. nov. for strain TP-A0598. The description is given below this section.
Analysis of 16S rDNA sequences is conventionally used as a primary method to identify strains at the genus level. Recently, the similarity of 99.0% was proposed as a boundary to discriminate species in actinobacteria classification [22]. However, a 16S rDNA sequence similarity of >99.0% does not necessarily guarantee that both strains belong to the same species. For example, we have recently classified two strains with completely identical 16S rDNA sequences into two different species [7]. In contrast, there are also cases in which strains showing a difference of 1 nt in their 16S rDNA sequence are classified into the same species [15]. These examples support the notion that 16S rDNA sequences do not have enough resolution to provide definitive identification [22]. For species-level identification, DNA–DNA relatedness provides a clear-cut criterion for bacterial classification [13,16]. We have investigated DNA–DNA relatedness based on whole genome sequences between Streptomyces strains with >99% similarities in 16S rDNA sequences [7,15,23,24]. The relationship between 16S rDNA sequence similarity and DNA–DNA relatedness is shown in Figure 3a. Proportional relation may be observed but the correlation coefficient is not high (R2 = 0.3935), and many strains (grey and red dots) are identified as different species even though their 16S rDNA sequence similarities are >99.0% [7,15,22].
In our previous reports [15,23,24,25], we introduced two hypotheses: (i) Strains belonging to the same species harbour very similar sets of smBGCs; (ii) Strains belonging to different species share only a limited number of smBGCs and each the strain has many species-specific ones. Hypothesis (ii) was supported by the present study even though the 16S rDNA sequence similarity between the two strains is 99.93%. We showed the relationship between DNA–DNA relatedness and the rate of PKS and NRPS gene clusters conserved between Streptomyces strains (RC) with >99.0% similarities in 16S rDNA sequences. The relationship is clearly proportional, with a correlation coefficient (R2) of 0.9481 (Figure 3b). A DNA–DNA relatedness value of 70% is the borderline level at which one can discriminate the same or different species in bacteria [17]. Strains within the same species (blue dot) do not show low RC values, whereas strains classified to different species do not show high RC values. These facts strongly support hypotheses (i) and (ii), described above, respectively. The idea that there is no relation between species classification and secondary metabolites is still widespread. There are two possible reasons why this idea has taken root and continues to persist: (a) only an (or a very limited number of) actually produced secondary metabolite has been investigated and genome information-based comprehensive analyses have not been conducted; (b) the classification of used strains is inaccurate and/or based on only 16S rDNA sequences [26,27,28,29,30,31,32]. If we had focused only on 16S rDNA sequence similarities, we would never have found out about the clear proportionality between phylogenetic distance and RC (Figure 3c). To obtain enough proof of concepts to verify our two hypotheses, we are continuing similar analyses on accurate classification and smBGCs for other phylogenetically close strains in the genus Streptomyces.
5. Description of S. lydicamycinicus sp. nov.
Streptomyces lydicamycinicus (lydi’ca.mi.ci’ni.cus. N. L. n. lydicamycin, an antibiotic; L. masc. suffix -icus adjectival suffix used with various meanings; N.L. masc. adj. lydicamycinicus related to lydicamycin, referring to the ability of the organism to produce lydicamycin and its congeners).
The data were taken from previous reports [5,6] and from this study. Aerobic and Gram stain-positive. Forms light yellow or vivid yellow substrate mycelia and white to grey aerial mycelia. Soluble pigment is yellow to reddish brown. Forms spiral spore chains and the spores were cylindrical, 0.5 × 0.9 μm in size, having a warty surface. Phenotypic characteristics when grown on agar media are shown in Table 1. Grows at 15–37 °C (optimal 25–28 °C), at pH 5–11 (optimal 6–9), and up to 5% NaCl (optimal 0%), but growths at 10°C and pH 11 are considered weak. d-Fructose, d-glucose, d-mannitol, d-raffinose, d-sucrose, and inositol are utilized as a sole carbon source. Assimilation of d-xylose and l-arabinose are weak. Produces acid from N-acethylglucosamine, d-adonitol, d-arabitol, d-fructose, d-galactose, gentiobiose, glycerol, glycogen, d-glucose, inositol, d-mannitol, d-mannose, d-melibiose, methyl-α-d-glucopyranoside, methyl-α-d-mannopyranoside, d-raffinose, d-ribose, d-sorbitol, starch, d-sucrose, and d-trehalose. Acid productions from amygdalin, arbutin, esculin ferric citrate, salicin, and xylitol are weak. Does not produce acid from the other sugars in API 50CH. In the API ZYM and API Coryne assays, N-acetylglucosaminidase, acid phosphatase, alkaline phosphatase, β-galactosidase, α-glucosidase, leucine aminopeptidase and phosphohydrolase are positive; N-acetyl-β-glucosaminidase, cystine aminopeptidase, esterase (C-4), esterase lipase (C-8), pyrrolidonyl arylamidase, and valine aminopeptidase are weakly positive; the other 11 enzymes are negative. Weakly ferments glucose, mannitol and ribose, but not glycogen, lactose, maltose, sucrose, and d-xylose. Negative for nitrate reduction. Hydrolyses gelatin but not esculin and urea. Catalase activity is positive. Oxidase activity is negative. Produces lydicamycin and TPU-0037-A to TPU-0037-D. The predominant menaquinones are MK-9(H6) and MK-9(H8); MK-9(H2) and MK-9(H4) are minor components. The major cellular fatty acids are C16:0, iso-C15:0, iso-C16:0, and anteiso-C15:0. The DNA G+C content of the type strain is 71.0 mol%.
The type strain is TP-A0598T (=NBRC 110027T), isolated from seawater collected in Namerikawa, Toyama, Japan.
Supplementary Materials
The following are available online at https://www.mdpi.com/2076-2607/8/3/370/s1, Table S1: Closest homolog of PKSs and NRPSs in the gene clusters of Streptomyces sp. TP-A0598, Table S2: Closest homolog of PKSs and NRPSs in the gene clusters of Streptomyces angustmyceticus NBRC 3934T.
Author Contributions
Conceptualization, supervision, project administration, visualization and writing—original draft preparation, H.K.; data curation, A.H.; investigation, H.K. and T.T.; resources, Y.I.; writing—review and editing, Y.I. and T.T. All authors have read and agree to the published version of the manuscript.
Funding
This research received no external funding.
Acknowledgments
We are grateful to Satomi Saitou for assistance of taxonomic experiments. We thank Yuko Kitahashi and Aya Uohara for finishing the genome sequences and annotating the NRPS and PKS genes and for registering the genome sequences in the DDBJ, respectively. The authors would like to thank Enago (www.enago.jp) for the English language review.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Phylogenetic trees of Streptomyces sp. TP-A0598 and type strains of the related species based on 16S rDNA sequences. (a) The neighbour-joining method; (b) the maximum likelihood method. Numbers on branches are the confidence limits estimated using bootstrap analysis with 1000 replicates; only values higher than 50% are at branching points. Strains with >99.0% similarities to Streptomyces sp. TP-A0598 are shown. The sequence of Embleya scabrispora NBRC 100760T (AB249946) was used as an outgroup. The accession numbers of Streptomyces strains are as follows: S. angustmycinicus NRRL B-2347T, MUAY01000275; S. tubercidicus DSM 40261T, AJ621612; S. libani subsp. libani NBRC 13452T, AB184414; S. libani subsp. libani NBRC 13452T, AB184414; S. catenulae NBRC 12848T, AB184191; S. catenulae NBRC 12848T, AB184191; S. hygroscopicus subsp. glebosus NBRC 13786T, AB184479; S. libani subsp. rufus LMG 20087T, AJ781351; S. platensis JCM 4662T, AB045882; S. decoyicus NRRL 2666T, LGUU01000106; S. sioyaensis NRRL B-5408T, DQ026654; S. chattanoogensis NRRL ISP-5002T, LGKG01000206; S. lydicus ATCC 25470T, RDTD01000009.
Figure 1.
Phylogenetic trees of Streptomyces sp. TP-A0598 and type strains of the related species based on 16S rDNA sequences. (a) The neighbour-joining method; (b) the maximum likelihood method. Numbers on branches are the confidence limits estimated using bootstrap analysis with 1000 replicates; only values higher than 50% are at branching points. Strains with >99.0% similarities to Streptomyces sp. TP-A0598 are shown. The sequence of Embleya scabrispora NBRC 100760T (AB249946) was used as an outgroup. The accession numbers of Streptomyces strains are as follows: S. angustmycinicus NRRL B-2347T, MUAY01000275; S. tubercidicus DSM 40261T, AJ621612; S. libani subsp. libani NBRC 13452T, AB184414; S. libani subsp. libani NBRC 13452T, AB184414; S. catenulae NBRC 12848T, AB184191; S. catenulae NBRC 12848T, AB184191; S. hygroscopicus subsp. glebosus NBRC 13786T, AB184479; S. libani subsp. rufus LMG 20087T, AJ781351; S. platensis JCM 4662T, AB045882; S. decoyicus NRRL 2666T, LGUU01000106; S. sioyaensis NRRL B-5408T, DQ026654; S. chattanoogensis NRRL ISP-5002T, LGKG01000206; S. lydicus ATCC 25470T, RDTD01000009.
Figure 2.
Venn diagram showing the putative compounds derived from PKS and NRPS pathways of phylogenetically close Streptomyces sp. TP-A0598 and Streptomyces angustmyceticus NBRC 3934T. Five are conserved between the two strains, whereas four and eight are specific to Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T, respectively. pk, moiety derived from PKS pathway; THN, tetrahydroxynaphthalene; x, unidentified amino acid; y, unknown building block; mx, methyl-amino acid; P, partial.
Figure 2.
Venn diagram showing the putative compounds derived from PKS and NRPS pathways of phylogenetically close Streptomyces sp. TP-A0598 and Streptomyces angustmyceticus NBRC 3934T. Five are conserved between the two strains, whereas four and eight are specific to Streptomyces sp. TP-A0598 and S. angustmyceticus NBRC 3934T, respectively. pk, moiety derived from PKS pathway; THN, tetrahydroxynaphthalene; x, unidentified amino acid; y, unknown building block; mx, methyl-amino acid; P, partial.
Figure 3.
Relationship among 16S rDNA sequence similarity, DNA–DNA relatedness, and rate of PKS and NRPS gene clusters conserved (RC) between Streptomyces strains. (a) Relationship between 16S rDNA sequence similarity and DNA–DNA relatedness; (b) Relationship between RC and DNA–DNA relatedness; (c) Relationship between RC and 16S rDNA sequence similarity. RC was calculated as stated in the Materials and Methods section. Blue, the same species (or the same genomospecies); red, different species with 16S rDNA sequence similarity of >99.9%; grey, different species with 16S rDNA sequence similarity of <99.5%. Data were taken from the following strain pairs: blue from Streptomyces sp. TP-A0882/Streptomyces diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces rubrogriseus NBRC 15455T/Streptomyces violaceoruber A3(2) [15], Streptomyces sp. NBRC 14016/Streptomyces sp. NBRC 13827, Streptomyces sp. NBRC 14016/Streptomyces sp. NBRC 13840 and Streptomyces sp. NBRC 13827/Streptomyces sp. NBRC 13840 [24]; red from Streptomyces sp. TP-A0598/S. angustmyceticus NBRC 3934T [this study] and Streptomyces sp. CHI39/S. fragilis NBRC 12862T [23]; grey from Streptomyces sp. TP-A0598/Streptomyces platensis DSM 40041T, S. angustmyceticus NBRC 3934T/S. platensis DSM 40041T [unpublished], Streptomyces sp. TP-A0882/Streptomyces coelicoflavus NBRC 15399T, Streptomyces sp. TP-A0882/S. rubrogriseus NBRC 15455T, S. rubrogriseus NBRC 15455T/S. coelicoflavus NBRC 15399T [15], Streptomyces sp. NBRC 14016/Streptomyces albospinus NBRC 13846T, Streptomyces sp. NBRC 13827/S. albospinus NBRC 13846T, and Streptomyces sp. NBRC 13840/S. albospinus NBRC 13846T [24].
Figure 3.
Relationship among 16S rDNA sequence similarity, DNA–DNA relatedness, and rate of PKS and NRPS gene clusters conserved (RC) between Streptomyces strains. (a) Relationship between 16S rDNA sequence similarity and DNA–DNA relatedness; (b) Relationship between RC and DNA–DNA relatedness; (c) Relationship between RC and 16S rDNA sequence similarity. RC was calculated as stated in the Materials and Methods section. Blue, the same species (or the same genomospecies); red, different species with 16S rDNA sequence similarity of >99.9%; grey, different species with 16S rDNA sequence similarity of <99.5%. Data were taken from the following strain pairs: blue from Streptomyces sp. TP-A0882/Streptomyces diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces rubrogriseus NBRC 15455T/Streptomyces violaceoruber A3(2) [15], Streptomyces sp. NBRC 14016/Streptomyces sp. NBRC 13827, Streptomyces sp. NBRC 14016/Streptomyces sp. NBRC 13840 and Streptomyces sp. NBRC 13827/Streptomyces sp. NBRC 13840 [24]; red from Streptomyces sp. TP-A0598/S. angustmyceticus NBRC 3934T [this study] and Streptomyces sp. CHI39/S. fragilis NBRC 12862T [23]; grey from Streptomyces sp. TP-A0598/Streptomyces platensis DSM 40041T, S. angustmyceticus NBRC 3934T/S. platensis DSM 40041T [unpublished], Streptomyces sp. TP-A0882/Streptomyces coelicoflavus NBRC 15399T, Streptomyces sp. TP-A0882/S. rubrogriseus NBRC 15455T, S. rubrogriseus NBRC 15455T/S. coelicoflavus NBRC 15399T [15], Streptomyces sp. NBRC 14016/Streptomyces albospinus NBRC 13846T, Streptomyces sp. NBRC 13827/S. albospinus NBRC 13846T, and Streptomyces sp. NBRC 13840/S. albospinus NBRC 13846T [24].
Table 1.
Cultural characteristics of Streptomyces sp. TP-0598 and the type strain of its closest phylogenetic neighbour.
Table 1.
Cultural characteristics of Streptomyces sp. TP-0598 and the type strain of its closest phylogenetic neighbour.
| Med. | Streptomyces sp. TP-A0598 | Streptomyces angustmyceticus NBRC 3934T | |
|---|---|---|---|
| ISP 2 | SM | ++, Vivid yellow | ++, Moderate yellow to moderate yellowish brown |
| AM | +, Light bluish grey | +, Yellowish grey to dark reddish brown | |
| SP | Moderate reddish brown | Moderate reddish brown | |
| ISP 3 | SM | +, Moderate yellowish brown to moderate reddish brown | ±, Moderate yellowish brown |
| AM | +, Light yellowish brown to dark greyish red | +, Light olive grey to olive grey | |
| SP | Light to moderate yellowish brown | Moderate reddish brown | |
| ISP 4 | SM | ±, Vivid yellow | ++, Pale yellow or moderate yellow |
| AM | +, Light bluish grey | +, Light olive grey to dark reddish brown | |
| SP | – | Moderate yellowish brown | |
| ISP 5 | SM | +, Light yellow | ++, Moderate yellow |
| AM | +, White | +, Light bluish grey | |
| SP | – | Moderate yellowish brown | |
| ISP 6 | SM | +, Pale yellow | +, Pale yellow |
| AM | – | +, White | |
| SP | – | – | |
| ISP 7 | SM | +, Pale yellow to moderate yellow | ++, Moderate yellow |
| AM | +, White to light bluish grey | +, White to light bluish grey | |
| SP | Moderate yellow | Moderate brown | |
| 266 | SM | +, Moderate yellow to strong brown | ++, Moderate yellowish brown to moderate reddish brown |
| AM | +, Light bluish grey | +, Light bluish grey | |
| SP | Deep orange | Moderate brown | |
| 228 | SM | +, Strong brown | ++, Moderate yellowish brown to moderate reddish brown |
| AM | +, Light bluish grey to light brownish grey | +, White to greyish blue | |
| SP | Deep orange | Moderate brown |
Med., agar medium; SM, substrate mycelium; AM, aerial mycelium; SP, soluble pigment; 266, Yeast extract-starch; 228, Bennett’s; –, not observed. Growth of mycelium was scored using ++ (good), + (normal) or ± (slight).
Table 2.
Characteristics different between Streptomyces sp. TP-A0598 and the type strain of its closest phylogenetic neighbour.
Table 2.
Characteristics different between Streptomyces sp. TP-A0598 and the type strain of its closest phylogenetic neighbour.
| Characteristic | Streptomyces sp. TP-A0598 | S. angustmyceticus NBRC 3934T |
|---|---|---|
| Morphological | ||
| Aerial mycelium * | White to grey | White to black |
| Substrate mycelium * | Light yellow or vivid yellow | Moderate yellow |
| Soluble pigment | Yellow to Reddish brown | – |
| Chemotaxonomic | ||
| Major fatty acid (%) | C16:0 (23), iso-C15:0 (12), iso-C16:0 (12), anteiso-C15:0 (11) | iso-C16:0 (31), anteiso-C15:0 (12), iso-C15:0 (9) |
| Physiological | ||
| Growth at/with: | ||
| 15 °C | + | w |
| 37 °C | +++ | + |
| pH 5 | w | + |
| pH 10 | + | +++ |
| pH 11 | w | +++ |
| 7%–15% NaCl | – | ++ or + |
| Gelatin hydrolysis | + | – |
| Acid production from: | ||
| d-Adonitol | + | – |
| Amygdalin | w | – |
| Arbutin | w | – |
| Esculin ferric citrate | w | – |
| Gentiobiose | + | – |
| Gluconate | – | + |
| d-Maltose | – | + |
| Methyl-α-d-mannopyranoside | + | – |
| Salicin | w | – |
| d-Turanose | – | + |
| Xylitol | w | + |
| d-Xylose | – | w |
| Biochemical | ||
| N-Acetyl-β-glucosaminidase | w | + |
| Chymotrypsin | – | w |
| β-Galactosidase | + | w |
| Pyrrolidonyl arylamidase | w | + |
* On Trypticase Soy agar. Differences on the other agar media are shown in Table 1; +, positive; –, negative; w, weak.
Table 3.
Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) in the gene clusters of Streptomyces sp. TP-A0598.
Table 3.
Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) in the gene clusters of Streptomyces sp. TP-A0598.
| Gene Cluster | Predicted Product | Locus Tag (TPA0598_) | Size (aa) | Domain Organization |
|---|---|---|---|---|
| t1pks-1 | Unknown | 10_00280 | 2436 | KS/AT/KR |
| 10_00270 | 1690 | KS/AT/DH/ACP | ||
| t1pks-2 | Unknown | 04_06320 | 444 | KS |
| 04_06310 | 2113 | KS/AT/DH/ER/KR/ACP | ||
| t2pks-1 | Spore pigment | 03_01500 | 422 | KSα |
| 03_01510 | 426 | KSβ (CLF) | ||
| 03_01520 | 89 | ACP | ||
| t2pks-2 (oxy) | Oxytetracycline | 07_00590 | 425 | KSα |
| 07_00600 | 426 | KSβ (CLF) | ||
| 07_00610 | 95 | ACP | ||
| t3pks-1 (rpp) | THN | 03_03810 | 353 | KS |
| nrps-1 | x-Leu-Val-y | 07_04820 | 1066 | C/A/T |
| 07_04810 | 641 | A(leu)/T | ||
| 07_04800 | 637 | A(val)/T | ||
| 07_04790 | 950 | C/T | ||
| nrps-2 | x-Asp-Asn-Leu-Phe-Thr-y-Leu | 02_01330 | 863 | C/A/T |
| 02_01450 | 2734 | A(asp)/T-C/A(asn)/T-C/A(leu)/T | ||
| 02_01460 | 2627 | C/A(phe)/T-C/A(thr)/T/E | ||
| 02_01470 | 1883 | C/T-C/A(leu)/T-TE | ||
| pks/nrps-1 | lydicamycin,TPU-0037-A to -D | 03_00740 | 3598 | KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP |
| 03_00750 | 7054 | KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP | ||
| 03_00760 | 3548 | KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP | ||
| 03_00770 | 1846 | KS/AT/DH/KR/ACP | ||
| 03_00780 | 5648 | KS/AT/DH/ER/KR/ACP -KS/AT/DH/KR/ACP -KS/AT/DH/KR/ACP | ||
| 03_00790 | 3662 | KS/AT/KR/ACP -KS/AT/DH/ER/KR/ACP | ||
| 03_00800 | 3265 | KS/AT/DH/KR/ACP -KS/AT/KR/ACP | ||
| 03_00820 | 1031 | C/A/T | ||
| 03_00840 | 1923 | ACP-KS/AT/DH/KR/ACP | ||
| pks/nrps-2 | Ser-y-Val-pk | 08_01960 | 556 | C/T |
| 08_01950 | 1139 | T-C/A(val)/T | ||
| 08_01940 | 1207 | KS/AT/ACP-TE | ||
| 08_01890 | 783 | A(ser)/T |
Gene clusters highlighted in boldface are also present in the genome of S. angustmyceticus NBRC 3934T. Abbreviations: A, adenylation; ACP, acyl carrier protein; AT, acyltransferase; C, condensation; DH, dehydratase; DHB, dihydroxybenzoate; E, epimerisation; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase; MT, methyltransferase; pk, moiety derived from PKS pathway; T, thiolation; 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 domains are shown in subscript brackets. The closest homolog of each protein is shown in Table S1.
Table 4.
PKS and NRPS in the gene clusters of S. angustmyceticus NBRC 3934T.
| Gene Cluster | Predicted Product | Locus Tag (San01_) | Size (aa) | Domain Organization |
|---|---|---|---|---|
| t1pks-1 | Unknown | 16600 | 2469 | KS/AT/KR |
| 16610 | 1601 | KS/AT/ACP | ||
| t1pks-2 | Unknown | 20810 | 428 | KS |
| 20820 | 2117 | KS/AT/DH/ER/KR/ACP | ||
| t1pks-3 (tsn) | Trichostatin A | s29-1 t,* | >375 | ACP/KS |
| RS35710 t | >471 | DH/ACP | ||
| RS35715 t | >897 | KS/AT | ||
| RS31690 t | >1500 | KS/AT/DH/KR/ACP | ||
| 64470 | 2005 | KS/AT/DH/KR/ACP-TE | ||
| t1pks-4 | Caniferolides | s04-1 t,* | >3397 | KS/AT/ACP -KS/AT/KR/ACP -KS/AT |
| RS35695 t | >990 | AT/KR/ACP | ||
| s40-2 t,* | >571 | KS | ||
| RS35360 t | >4323 | AT/KR/ACP -KS/AT/KR/ACP -KS/AT/DH/KR/ACP | ||
| 71810 | 3953 | KS/AT/DH/KR/ACP -KS/AT/DH/ER/KR/ACP | ||
| RS35370 t | >2135 | KS/AT/KR/ACP -KS | ||
| RS35705 t | >1815 | AT/DH/ER/DH/KR/ACP -KS | ||
| RS35690 t | >1167 | AT/KR/ACP | ||
| s39-1 t,* | >577 | KS | ||
| RS35595 t | >6115 | AT/KR/ACP -KS/AT/KR/ACP -KS/AT/DH/ACP -KS/AT/KR/ACP -KS | ||
| RS31970t | >1754 | AT/DH/ER/KR/ACP | ||
| 65020 | 5281 | KS/AT/KR/ACP -KS/AT/DH/ER/KR/ACP -KS/AT/KR/ACP | ||
| 65010 | 3643 | KS/AT/KR/ACP -KS/AT/DH/KR/ACP -TE | ||
| 64920 | 2404 | CoL/T-KS/AT/DH/KR/ACP | ||
| t2pks-1 | Spore pigment | 26680 | 422 | KSα |
| 26670 | 416 | KSβ (CLF) | ||
| 26660 | 96 | ACP | ||
| t2pks-3 | Trioxacarcin | 00550 | 421 | KSα |
| 00560 | 417 | KSβ (CLF) | ||
| 00570 | 89 | ACP | ||
| 00580 | 660 | AT | ||
| t3pks-1 (rpp) | THN | 24600 | 354 | KS |
| nrps-1 | x-Leu-Val-y | 06160 | 1066 | C/A/T |
| 06150 | 641 | A(leu)/T | ||
| 06140 | 637 | A(val)/T | ||
| 06130 | 950 | C/T | ||
| nrps-3 | Leu-Ala-x-Ala-Leu-x-Thr -Orn-x-Leu | 24170 | 6209 | A(leu)/T-C/A(ala)/T/E -C/A/T/E-C/A(ala)/T/E-C/A(leu)/T |
| 24160 | 6677 | C/A/T/E-C/A(thr)/T-C/A(orn)/T/E -C/A/T-C/A(leu)/T/E | ||
| nrps-4 | mx-x-Gly- | 36160 | 3675 | A/MT/T-C/A/T-C/A(gly)/T |
| s07-1 t,* | >398 | C | ||
| nrps-5 | Thr-Thr-x, Arg-Pro | 27520 t | >3401 | A(thr)/T-C/A(thr)/T-C/A/T-TE |
| 27580 | 591 | A(arg)/T | ||
| 27590 | 1,446 | C/A(pro)/T-TD | ||
| pks/nrps-3 | Guadinomine, partial | 61570 | 252 | KR |
| 61560 | 303 | AT | ||
| 61550 | 302 | AT | ||
| 61530 | 2393 | A(pip)/T-KS/AT/DH/KR/ACP | ||
| 61520 | 270 | TE | ||
| 61190 | 1178 | A/T-TD | ||
| pks/nrps-4 | Ala-Thr-pk | 12760 | 93 | ACP |
| 12770 | 674 | KS/DH | ||
| 12780 | 526 | A(ala) | ||
| 12800 | 1050 | A/T(thr)-C | ||
| 12810 | 274 | TE |
* Italic, locus tag is not provided. The open reading frame (ORF) regions in nucleotides are as follows: s29-1t, BLAG01000032 1-1,125 (+); s04-1t, BLAG01000007 3-10,193 (-); s40-2t, BLAG01000043 3,024-4,736 (+); s39-1t, BLAG01000042 1-1,734 (-); s07-1t, BLAG01000010 2-1,195 (-). Gene clusters highlighted in boldface are also present in the genome of Streptomyces sp. TP-A0598. t Not completely sequenced because the ORF is present at the terminal of the scaffold sequence. Undetermined domains are shown as ‘…’. Abbreviations are the same as those of Table 3. mx, methyl-amino acid; TD, termination domain. The closest homolog of each protein is shown in Table S2.
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Komaki, H.; Hosoyama, A.; Igarashi, Y.; Tamura, T. Streptomyces lydicamycinicus sp. nov. and Its Secondary Metabolite Biosynthetic Gene Clusters for Polyketide and Nonribosomal Peptide Compounds. Microorganisms 2020, 8, 370. https://doi.org/10.3390/microorganisms8030370
AMA Style
Komaki H, Hosoyama A, Igarashi Y, Tamura T. Streptomyces lydicamycinicus sp. nov. and Its Secondary Metabolite Biosynthetic Gene Clusters for Polyketide and Nonribosomal Peptide Compounds. Microorganisms. 2020; 8(3):370. https://doi.org/10.3390/microorganisms8030370
Chicago/Turabian StyleKomaki, Hisayuki, Akira Hosoyama, Yasuhiro Igarashi, and Tomohiko Tamura. 2020. "Streptomyces lydicamycinicus sp. nov. and Its Secondary Metabolite Biosynthetic Gene Clusters for Polyketide and Nonribosomal Peptide Compounds" Microorganisms 8, no. 3: 370. https://doi.org/10.3390/microorganisms8030370
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