Open Access

Activation of a plasmid-situated type III PKS gene cluster by deletion of a wbl gene in deepsea-derived Streptomyces somaliensis SCSIO ZH66

  • Huiming Huang1,
  • Lukuan Hou1,
  • Huayue Li1,
  • Yanhong Qiu1,
  • Jianhua Ju2 and
  • Wenli Li1Email author
Microbial Cell Factories201615:116

https://doi.org/10.1186/s12934-016-0515-6

Received: 1 March 2016

Accepted: 17 June 2016

Published: 27 June 2016

Abstract

Background

Actinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblA so gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66.

Results

wblA so from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblA so mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblA so in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66.

Conclusions

wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.

Keywords

Deepsea-derived Streptomyces Cryptic gene cluster whiB-like (wbl) gene Violapyrones (VLPs) Type III polyketide synthase (PKS)

Background

Given marine environmental conditions are extremely different from the terrestrial environment, marine actinomycete strains have become an important source of pharmacologically active compounds [1]. Recently, microbial genome sequencing has brought to light a large number of cryptic secondary metabolite biosynthetic gene clusters, demonstrating the tremendous genetic potentials for producing secondary metabolites, which fundamentally refreshed the way for natural product discovery [2, 3]. However, most of these biosynthetic pathways are not expressed or only expressed in a very low titer under ordinary laboratory conditions, which severely hampered the discovery of bioactive compounds. Thus activation of cryptic gene clusters has become a tempting and rapidly developing field [46].

The control of antibiotic biosynthesis involves complex regulatory cascades and intertwined networks, and many of them are coordinately regulated together with the morphological differentiation [5, 7]. In general, regulators are classified as pathway-specific regulator (or cluster-situated regulator) and global regulator (or pleiotropic regulator) per their modes of action [5]. Among them, the whiB-like (wbl) regulatory genes have received much attention due to their diverse biological roles, such as in morphological differentiation and secondary metabolism [8, 9]. Wbls are small cytoplasmic proteins confined to actinobacteria, which contain four conserved cysteine residues coordinating an Fe-S cluster [8, 10]. The chromosome of Streptomyces coelicolor A3(2) contains 11 wbl genes: whiB and whiD genes are involved in sporulation; wblA controls major development transitions; wblC mutant was hypersensitive to a wide range of antibiotics; inactivation of wblE was lethal; no obvious effects were observed when the other six wbl genes (wblH, -I, -J, -K, -L, -M) were inactivated [8]. Notably, wblA and its homologues have been reported to serve as global regulators for the biosynthesis of various antibiotics, mostly in a negative manner (such as for actinorhodin [8], tautomycetin [11] and doxorubicin [12]); conversely, it was found to exert dual function in antibiotic biosynthesis in Streptomyces chattanoogensis L10 [13] and Streptomyces ansochromogenes 7100 [14] as well.

In our efforts to discover novel natural products from marine Streptomyces strains by using genome mining strategy, wblA so gene was set as the target to activate cryptic gene clusters in the deepsea-derived Streptomyces somaliensis SCSIO ZH66, leading to significant changes of secondary metabolites production in the ΔwblA so mutant, from which α-pyrone compound violapyrone B (VLP B, 1, Fig. 1) was isolated and identified. VLPs were first isolated from Streptomyces violascens YIM 100525 obtained from Hylobates hoolock feces, and were reported to inhibit the growth of Bacillus subtilis and Staphylococcus aureus [15]. Simultaneously, VLPs were also named as presulficidins, which serve as sulfate donors relaying sulfonate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to caprazamycin [16].
Fig. 1

The chemical structures of violapyrones (VLPs 1-5). 1, VLP B; 2, VLP A; 3, VLP J; 4, VLP C; 5, VLP H. 3 is a novel VLP analogue isolated from the ΔvioB mutant

Pyrones are usually assembled by type III polyketide synthases (PKSs) [1620], which are homodimeric ketosynthases that catalyze condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions [21]. Notably, type III PKSs usually exhibit broad substrate promiscuity, and can recognize unnatural substrates to generate novel unnatural products, which renders them excellent candidates for enzymatic engineering to expand chemical space of polyketides [22, 23]. Several pyrone-encoding type III PKS have been identified. For instance, BpsA synthesizes of triketide pyrones from long-chain fatty acyl-CoA thioesters as starter substrates and malonyl-CoA as extender substrate in B. subtilis [17]; Gcs catalyzes the condensation of an acyl carrier protein (ACP) ester of β-keto acid and ethylmalonyl-CoA to form germicidins in S. coelicolor A3(2) [18, 23]; DpyA catalyzes the synthesis of alkyldihydropyrones using β-hydroxyl acid thioesters as starter substrates in Streptomyces reveromyceticus [19]; ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate to give corresponding alkylpyrones [20]; Cpz6 was demonstrated to encode presulficidins in vivo putatively from CoA- or ACP-activated iso-acyl starter units as well as one malonyl and one methylmalonyl unit in Streptomyces sp. MK730–62F2 [16].

Herein, VLP B (1) was found via inactivation of the global regulatory gene wblA so from deepsea-derived S. somaliensis SCSIO ZH66; the type III PKS gene vioA encoding VLP was then identified; subsequently, the VLP biosynthetic gene cluster was characterized, leading to isolation of another four VLPs compounds (25), among which one was new (3) and notably two (4 and 5) exhibited improved anti-MRSA (methicillin-resistant S. aureus, MRSA) activity than 1.

Results

Inactivation of wblA so led to significant enhancement of VLP B production

As wblA and its orthologous genes were reported to be down-regulators for secondary metabolism in Streptomyces strains [11, 12, 24, 25], the orthologous gene designated wblA so was probed from S. somaliensis SCSIO ZH66 and was set as a target for activating cryptic gene clusters. WblAso exhibited 85 % identity to WblA (CAB43030.1) from S. coelicolor A3(2) with a highly conserved Cys-X21-Cys-X2-Cys-X5-Cys motif, which coordinate a redox–sensitive iron-sulphur cluster [10]. Gene inactivation was performed to detect its impact on production of secondary metabolites in S. somaliensis SCSIO ZH66. The ΔwblA so mutant was obtained as described in the “Methods” section. After confirmation by PCR analysis (Additional file 1: Figure S1), fermentations were carried out and the accumulated metabolites were analyzed by high-pressure liquid chromatography (HPLC). As shown in Fig. 2a, the profile of the ΔwblA so mutant (panel ii) substantially changed and productions were enhanced compared to those in the wild-type strain (panel i), indicating that WblAso functions as a global negative regulator for secondary metabolism in S. somaliensis SCSIO ZH66. Compound 1 at retention time of 33.1 min, which was a significantly enhanced peak (by about fivefold) at wavelength of 290 nm, was then purified and subject to structural analysis. The UV spectrum of 1 displayed λmax at 290 nm (Additional file 1: Figure S2A), and the chemical formula of 1 was determined to be C13H2O3 by high-resolution electrospray mass spectrometry (HR-ESI–MS) (m/z 225.1483 [M + H]+, calcd 225.1491) (Additional file 1: Figure S2B). The 1H NMR data of 1 was further recorded (Additional file 1: Figure S2C), leading to its identification as antibiotic VLP B (Fig. 1) by comparison of all the above data with those previously reported [15]. VLP B was also named as presulficidin A, which relays sulfonate from PAPS to caprazamycin [16].
Fig. 2

Comparisons of the wild-type strain and the mutant strain ΔwblA so . a HPLC traces of the fermentation broths from the wild-type strain (i) and the mutant strain ΔwblA so (ii). “1″ indicates the accumulated compound VLP B by ΔwblA so . b The scanning electron micrographs of the wild-type strain (i) and the mutant strain ΔwblA so (ii) after incubation on MS plate at 30 °C for 4 days

Identification of the type III PKS gene involved in VLP B biosynthesis

To identify the VLP biosynthetic gene cluster, bioinformatic analysis of the S. somaliensis SCSIO ZH66 genome was performed, revealing the presence of two type III PKS genes, pksIII-1 and pksIII-2. BlastP searches against the GenBank nr database revealed that PksIII-1 exhibited 72 % identity to the 1,3,6,8-tetrahydroxynaphthaene synthase (THNS) from S. coelicolor A3(2) (WP_011027653.1), and PksIII-2 displayed highly homology (95 % identity) to a putative THNS from Streptomyces sp. W9 (WP_012840496.1, Table 1). Further sequence alignment of PksIII-2 with reported pyrone synthases indicated that PksIII-2 exhibited 53 % identity to Cpz6 and 36 % identity to DpyA (BAQ19510.1), suggesting PksIII-2 might encode VLP B in S. somaliensis SCSIO ZH66. Both pksIII-1 and pksIII-2 were inactivated, resulting in ΔpksIII-1 and ΔpksIII-2 mutants (Additional file 1: Figure S3 and S4). HPLC analysis of their fermentation products showed that ΔpksIII-2 failed to accumulate 1 (Fig. 3 panel iii), while the ΔpksIII-1 mutant (Fig. 3 panel ii) produced the same little amount of 1 as that of the wild type strain (Fig. 3 panel i). This result demonstrated that it is pksIII-2 that is involved in VLP B biosynthesis, and thus, pksIII-2 was renamed as vioA.
Table 1

Proposed functions of proteins encoded by the vio biosynthetic gene cluster in S. somaliensis SCSIO ZH66

Protein

Size (aa)

Proposed function

Homologs

Protein/organism

Accession no. (identity/similarity %)

Orf(-3)

88

Hypothetical protein

pCQ3_109/Streptomyces sp. W9

WP_012840495.1 (65/70)

Orf(-2)

63

Hypothetical protein

SVEN_2103/Streptomyces venezuelae ATCC 10712

WP_015033307.1 (60/66)

Orf(-1)

70

Hypothetical protein

pCQ4.14/Streptomyces sp. W75

WP_015060953.1 (50/52)

VioA

350

Type III PKS

pCQ3.110c/Streptomyces sp. W9

WP_012840496.1 (95/97)

VioB

272

XRE-family regulator

pCQ4.19c/Streptomyces sp. W75

WP_015060958.1 (95/95)

Orf1

158

Hypothetical protein

pCQ3.4c/Streptomyces sp. W9

WP_012840390.1 (61/62)

Orf2

76

Hypothetical protein

pCQ4.20c/Streptomyces sp. W75

WP_015060959.1 (31/34)

Fig. 3

HPLC traces of the fermentation broths from S. somaliensis SCSIO ZH66 strains. (i) the wild-type strain; (ii) ΔpksIII-1; (iii) ΔvioA; (iv) ΔvioB; (v) Δorf1; (vi) Δorf(-1-2)

Characterization of the vio gene cluster

We then analyzed the surrounding sequences of vioA, and found that this gene is situated in a circular plasmid with the size of ~85 kb. Adjacent to vioA, a regulatory gene vioB was found, which displayed 94 % identity to an unknown regulator from Streptomyces sp. W9 (WP_012840387.1); Orf1-Orf2 and Orf(-1)-Orf(-3) are hypothetical proteins with unknown functions (Fig. 4, Table 1). Gene inactivation was performed to investigate their functions (Additional file 1: Figure S5–S7). As shown in Fig. 3, inactivation of vioB led to production enhancement of 1 by about eightfold (panel iv), suggesting it serves as a negative regulator; while inactivation of orf1 (panel v) and orf(-1-2) (panel vi) had no obvious impacts on 1 production, indicating that they are probably beyond the gene cluster. Therefore, the vio gene cluster consists of only two genes, the structural gene vioA and the regulatory gene vioB.
Fig. 4

Genetic organization of the vio gene cluster. Proposed functions of individual open reading frames are coded with various patterns and summarized in Table 1

Identification of VLP analogues with improved anti-MRSA activity

Careful analysis of the ΔvioB mutant revealed that a few more VLP analogues were accumulated as well in addition to 1 (Fig. 3, panel iv). Therefore, large scale fermentations were performed, leading to isolation of another 4 VLPs (25) (Fig. 1). The molecular formula of 3 was C12H18O3, as determined by HR-ESI–MS (m/z 211.1328 [M + H]+, calcd 211.1334) (Additional file 1: Figure S8A), having less CH2 unit than that of 1. Full sets of 1D and 2D NMR spectra of 3 were acquired, thereby allowing us to complete its structure assignments (Table 2; Fig. 5a, Additional file 1: Figure S8). According to the COSY and HMBC correlations, 3 has the same 3-methyl-4-hydroxy-α-pyrone backbone as that in 1, and the side chain at C-6 was assigned as hexyl (Fig. 5a). Thus, 3 was identified to be a novel VLP analogue, named VLP J (Fig. 1). Compounds 2, 4 and 5 were identified as VLP A, VLP C and VLP H, respectively, by comparison of their HR-ESI–MS and 1H NMR data with those of reported (Additional file 1: Figure S9–S11) [15, 26]. Inspection of the structure of VLPs 15 suggests that they are probably assembled from different CoA- or ACP-tethered β-keto acids from branched-chain (for 1, 2, 4 and 5) or straight-chain (for 3) fatty acid metabolism and methylmalonyl CoA via Claisen condensation.
Table 2

Assignments from 500 MHz NMR spectroscopies of 3 in DMSO-d 6

Position

Compound 3

δH (J in Hz)

δC

1

165.2

2

95.5

3

1.71 (s)

8.3

4

166.7

5

5.91 (bs)

100.2

6

161.9

7

2.37 (t, 7.5)

32.6

8

1.51 (m)

26.1

9

1.27 (m)

27.6

10

1.26 (m)

30.9

11

1.26 (m)

21.7

12

0.85 (t, 6.5)

13.7

Fig. 5

Correlations of compound 3 and bioassays of VLPs. a Key HMBC and COSY correlations of 3 in DMSO-d6. b Antibacterial activity of VLPs against MRSA. 100 µg of each compound dissolved in methanol was added. Inhibition zones were observed after incubation at 37 °C for 20 h; methanol has no impact on the growth of MRSA

Furthermore, the anti-MRSA bioactivities of VLPs 15 were investigated, and the result revealed that all of them with the exception of 2 showed inhibition against MRSA at the concentration of 100 μg/well; compound 5 with a minimum inhibitory concentration (MIC) value of 25 μg/mL gave the best activity among the VLPs tested (Fig. 5b, Additional file 1: Table S1). These results demonstrated that the polarity of the VLPs, which are mostly up to the length of alkyl side chains, plays an essential role for their anti-MRSA activity.

Effects of wblA so gene inactivation on the vio gene cluster

To detect the effects of wblA so on the expression of the vio gene cluster, the transcription levels of vioA and vioB were analyzed by quantitative real-time RT-PCR (qPCR). As shown in Fig. 6a, transcription of vioB was substantially reduced in the ΔwblA so mutant in comparison to the wild-type strain during fermentation; simultaneously, expression of vioA was obviously enhanced, suggesting that activation of the vio gene cluster was probably achieved via repression of vioB. We next set out to determine whether WblAso regulated the vio gene cluster directly. To this end, we performed electrophoretic mobility shift assay (EMSA) to detect the binding ability of WblAso to the promoter regions of vioA and vioB under anaerobic conditions; however, no binding was observed (data not shown).
Fig. 6

Effects of wblA so inactivation on the expression levels of the vio (a), whi (b) and wbl (c) genes. For the vio genes, the transcription levels were detected at 3, 5 and 7 days in the wild-type strain and the mutant strain ΔwblA so cultured in fermentation medium at 30 °C. For the whi and wbl genes, the transcription levels were detected in the wild-type strain and the ΔwblA so mutant grown on MS plates at 30 °C for 4 days. The transcription level of hrdB was used as an internal control. Error bars indicated standard deviations (n = 3)

Effects of wblA so inactivation on morphology of S. somaliensis SCSIO ZH66

Moreover, the effects of wblA so inactivation on morphological development were investigated, and the phenotype of the ΔwblA so mutant was compared to the wild-type strain on mannitol-soy flour (MS) medium. While the wild-type strain sporulated well when incubated on MS plate at 30 °C for 4 days, the ΔwblA so mutant defected in sporulation at the same conditions (Additional file 1: Figure S12). Scanning electron microscopy of the surfaces of the strains revealed abundant spore chains of the wild-type strain (Fig. 2b, panel i), in contrast, thin and sparse aerial hyphae of the ΔwblA so mutant (Fig. 2b, panel ii), consistent with previous observations in other Streptomyces strains [8, 13, 14]. We further evaluated the transcription levels of whi genes as well as other wbl genes. As shown in Fig. 6b, the transcription of whiD and whiH was almost abolished in the ΔwblA so mutant; the transcription levels of whiG and whiI were severely decreased to ~6 % of the wild-type levels; conversely, only slight difference were observed for the transcription levels of whiA (~85 % of the wild-type level) and whiB (~82 % of the wild-type level). Interestingly, the transcription levels of the other wbl genes (wblC, wblE, wblH, wblI and wblK) were also significantly decreased to ~5–55 % of the wild-type levels (Fig. 6c). These findings implied that wblA so served as a multifunctional regulator via a very complex network involved in whi genes as well as other wbl genes.

Discussion

Manipulation of global regulators was one of the effective strategies for activation of cryptic secondary metabolite biosynthetic gene clusters [5, 6]. However, the orthologues of a specific regulator can play distinct roles in different biological backgrounds. Marine microorganisms have been endowed with unique physiological functions, and thereby unusual metabolic pathways during evolution in specific ecological environments [1], indicating their underlying regulation mechanisms might be unique as well. In the present study, the global regulatory gene wblA so was discerned from deepsea-derived S. somaliensis SCSIO ZH66, and was then deleted to activate cryptic gene clusters, leading to identification of anti-MRSA compound VLP B and thereafter its encoding gene cluster.

So far, the regulatory mechanisms of WblA and its orthologues in antibiotics biosynthesis are still unknown [13, 14]. WblAs all harbor a conserved helix-turn-helix DNA-binding motif, indicating they probably function by binding the promotor regions of the target genes. However, no evidences have been obtained to support their binding abilities to antibiotic biosynthetic genes [13]. Further efforts need to be devoted to clarify their mechanisms executing regulation of antibiotics biosynthesis. In Streptomyces species, production of secondary metabolites is closely coordinated with morphological differentiation [5, 7]. Inactivation of wblA and its orthologous genes had effects on both processes [8, 13, 14], and hence the transcription levels of whi genes as well as other wbl genes were investigated in this study (Fig. 6). Interestingly, transcription evaluation of the whi genes in the ΔwblA ch mutant suggested whiB, whiH and whiI were nearly not transcripted; the expression of whiA was decreased to about ~20 % of the wild-type level; on the contrary, the transcription level of whiD was increased slightly [13]. However, our result revealed that transcription levels of not only whiH and whiI but also whiD and whiG were all severely decreased in the ΔwblA so mutant compared to those in the wild-type strain; conversely, the expression of whiA and whiB was only decreased slightly (Fig. 6b). The different impacts caused by inactivation of wblA orthologues probably implied their varied regulation mechanisms in different strains. It is worth to mention that the transcription levels of other wbl genes in the ΔwblA so mutant were also decreased by different degrees (Fig. 6c), suggesting wblA so probably interacts with other wbl genes as well. Thus, we could speculate that wblA so is a multifunctional regulator with an intertwined and sophisticated mechanism.

Although both Cpz6 and VioA catalyze the formation of presulficidin A/VLP B, the genetic contexts of cpz6 and vioA are totally different: cpz6 is situated in the caprazamycin biosynthetic gene cluster, and vioA lies in the vio gene cluster consisting of only two genes; in addition, VioA encodes VLPs with different types and ratios from presulficidins synthesized by Cpz6 [16]. These facts suggest VLPs might serve different biological functions in S. somaliensis SCSIO ZH66 from presulficidins in Streptomyces sp. MK730–62F2, which relay sulfonate from PAPS to caprazamycin [16]. The different types and ratios of the products encoded by Cpz6 and VioA might be dictated by their different substrate preference as well as substrate availability in different biological backgrounds.

Here, for the first time, VLPs were shown to display anti-MRSA activity (Fig. 5b). As compounds 15 all have the same 3-methyl-4-hydroxy-α-pyrone backbone, the differences in their anti-MRSA activity can be ascribed to the influence of the alkyl side chain at C-6 (Fig. 1). As shown in Fig. 5b, the anti-MRSA activity increased with decrease in the polarity of the compounds, suggesting that the lipophilic nature of the alkyl chain plays an important role for the activity. These findings pointed out prospective directions for bioactivity improvement of VLPs. Further exploration of substrate promiscuity of VioA towards unnatural malonyl-CoA analogues would provide more opportunities to engineer chemical diverse polyketides using rational approaches.

Conclusions

A plasmid-situated type III PKS gene cluster was activated by deletion of the wblA so gene in deepsea-derived S. somaliensis SCSIO ZH66, leading to isolation of anti-MRSA α-pyrone compound 1. Further identification and characterization of the vio gene cluster resulted in one novel VLP analogue (3) and two VLPs analogues (4 and 5) with improved anti-MRSA bioactivity than that of 1. Therefore, wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains. In addition, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing additional opportunities to biosynthetically engineer chemical diverse polyketides for drug development.

Methods

Bacterial strains, plasmids, and culture conditions

All strains and plasmids used in this study are listed in Additional file 1: Table S2. Escherichia coli DH5α was served as the host for general subcloning [27]. Escherichia coli Top10 (Invitrogen, Carlsbad, La Jolla, CA, USA) was used as the transduction host for cosmid library construction. Escherichia coli ET12567/pUZ8002 [28] was used as the cosmid donor host for E. coli-Streptomyces intergenic conjugation. Escherichia coli BW25113/pIJ790 was used for λRED-mediated PCR-targeting [29]. The S. somaliensis SCSIO ZH66 (CGMCC NO. 9492) was isolated from the deep sea sediment collected at a depth of 3536 meters of the South China Sea (120° 0.250′E; 20° 22.971′N), and has been described previously [30]. E. coli strains were routinely cultured in Luria–Bertani (LB) liquid medium at 37 °C, 200 rpm, or LB agar plate at 37 °C. Streptomyces strains were grown at 30 °C on MS medium for sporulation and conjugation, and were cultured in tryptic soy broth (TSB) medium for genomic DNA preparation. Fermentation medium consists of 1 % soluble starch, 2 % glucose, 4 % corn syrup, 1 % yeast extract, 0.3 % beef extract, 0.05 % MgSO4·7H2O, 0.05 % KH2PO4, 0.2 % CaCO3, and 3 % sea salt, pH = 7.0, which was further supplemented with 1.5 % XAD-16 resin when fermenting the ΔwblA so mutant.

DNA isolation and manipulation

Plasmid extractions and DNA purifications were carried out using standardized commercial kits (OMEGA, Bio-Tek, Guangzhou, China). PCR reactions were carried out using Pfu DNA polymerase (TIANGEN, Beijing, China). Oligonucleotide synthesis and DNA sequencing were performed by Sunny Biotech company (Shanghai, China). Restriction endonucleases and T4 DNA ligase were purchased from Fermentas (Shenzhen, China).

Genomic library construction and library screening

S. somaliensis SCSIO ZH66 genomic DNA was partially digested with Sau3AI, and fragments with the size of 40-50 kb were recovered and dephosphorylated with CIAP, and then ligated into SuperCos1 that was pretreated with XbaI, dephosphorylated, and digested with BamHI. The ligation product was packaged into lambda particles with the MaxPlax Lambda Packaging Extract (Epicenter, Madison, WI, USA) as per the manufacture’s instruction and plated on E. coli Top10. The titer of the primary library was about 2 × 106 cfu per μg of DNA. Specific primers were designed per the draft genome sequence for library screening against 2500 colonies by PCR (Additional file 1: Table S3).

Sequence analysis

The two type III PKSs were identified from the S. somaliensis SCSIO ZH66 genome using the antiSMASH program [31]. orf assignments and their proposed function were accomplished by using the FramePlot 4.0beta (http://nocardia.nih.go.jp/fp4) [32] and Blast programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [33], respectively.

Gene inactivation

Gene inactivation in S. somaliensis SCSIO ZH66 was performed using the REDIRECT Technology according to the literature protocol [29, 34]. The amplified aac(3)IV-oriT resistance cassette from pIJ773 was transformed into E. coli BW25113/pIJ790 containing corresponding cosmid to replace an internal region of the target gene. Mutant cosmids were constructed (Additional file 1: Table S4) and introduced into S. somaliensis SCSIO ZH66 by conjugation from E. coli ET12567/pUZ8002 according to the reported procedure [35]. The desired mutants were selected by the apramycin-resistant and kanamycin-sensitive phenotype, and were further confirmed by PCR (Additional file 1: Table S5, Figures. S1, S3–S7).

Production and analyses of VLPs

The fermentation cultures were harvested by centrifugation, and the supernatant was extracted twice with an equal volume of ethyl acetate. The combined EtOAc extracts were concentrated in vacuo to afford residue A. In the case of the ΔwblA so mutant, the precipitated mycelia and XAD-16 resin were extracted twice with acetone. The extracts were combined, and acetone was evaporated in vacuo to yield residue B. The combined residues (for the ΔwblA so mutant) or residue A (for the other mutants) were dissolved in MeOH, filtered through a 0.2 μm filter, and subject to HPLC. The HPLC system consisted of Agilent 1260 Infinity Quaternary pumps and a 1260 Infinity diode-array detector. Analytical HPLC was performed on an Eclipse C18 column (5 μm, 4.6 × 150 mm) developed with a linear gradient from 5 to 80 % B/A in 40 min (for analyzing ΔwblA so mutant) or 20 to 70 % B/A in 20 min (for analyzing all the other mutants reported here) (phase A: 0.1 % formic acid in H2O; phase B: 100 % acetonitrile supplemented with 0.1 % formic acid) followed by an additional 10 min at 100 % B at flow rate of 1 mL/min and UV detection at 290 nm. For VLPs purification, semi-preparative HPLC was carried out using an YMC-Pack ODS-A C18 column (5 μm, 120 nm, 250 × 10 mm). Samples were eluted with a linear gradient from 50 to 80 % B/A in 40 min, followed by 100 % B for 10 min at a flow rate of 2.0 mL/min and UV detection at 290 nm. The identity of VLPs were confirmed by HR-ESI–MS and NMR analysis. HR-ESI-MS was carried out on Thermo LTQ-XL mass spectrometer. NMR data was recorded with an Agilent-DD2 500 spectrometer.

Microscopy

For scanning electron microscopy, colonies were fixed in 2.5 % (v/v) glutaraldehyde at 4 °C overnight, stained with osmic acid for 2–4 h and dehydrated with ethanol at different concentrations. Each sample was coated with platinum-gold and then detected using a Hitachi S-4800 scanning microscope.

Biological assays

The antibacterial activity of VLPs was assayed by agar diffusion test against methicillin-resistant S. aureus CCARM 3090. The MRSA strain was seeded in LB medium and then incubated at 37 °C for 20 h. After dilution with LB to 108 cfu/mL, 25 μL of cell suspension was mixed with 25 mL LB medium for each plate. Subsequently, 10 μL of VLPs, at a final concentration of 10 mg/mL, were added to the sample wells and the inhibition zones were observed after incubation at 37 °C for 20 h. For determination of MIC values, the VLPs solutions were prepared in methanol and dispensed into 96-well plates using serial dilution method. Different concentration ranges were used for each compound. The overnight culture of MRSA was diluted to 106 cfu/mL when used. LB broth was used as a blank control, and methanol and tetracycline were used as a negative control and a positive control, respectively. The growth of MRSA was measured after 12 h of incubation at 30 °C on a microplate reader (Epoch2, Biotech) at wavelength of 600 nm. Each assay was performed in triplicate.

Transcriptional analysis by quantitative real-time RT-PCR

Total RNAs were prepared using Ultrapure RNA Kit (CWBio. Inc., Beijing, China). qPCR was performed as described previously [30]. The primers for qPCR are listed in Table S6.

Nucleotide sequence accession number

The nucleotide sequences of wblA so , pksIII-1 and the vio gene cluster reported in this paper have been deposited in the GenBank database under accession numbers of KU534996, KU534994 and KU534995, respectively.

Abbreviations

wbl

whiB-like

VLP: 

violapyrone

MRSA: 

methicillin-resistant Staphylococcus aureus

PKS: 

polyketide synthase

PAPS: 

3′-phosphoadenosine 5′-phosphosulfate

ACP: 

acyl carrier protein

HPLC: 

high-pressure liquid chromatography

HR-ESI–MS: 

high-resolution electrospray mass spectrometry

THNS: 

1,3,6,8-tetrahydroxynaphthaene synthase

MIC: 

minimum inhibitory concentration

qPCR: 

quantitative real-time RT-PCR

EMSA: 

electrophoretic mobility shift assay

MS: 

mannitol-soy flour

LB: 

Luria–Bertani

TSB: 

tryptic soy broth

Declarations

Authors’ contributions

HH and LH performed the experiments and wrote the draft manuscript. HL was involved in NMR analysis. YQ assisted in transcriptional analysis. JJ sequenced the vio gene cluster. WL supervised the whole work and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to Prof. Huarong Tan (Institute of Microbiology, Chinese Academy of Sciences, China) for kindly providing us Supercos1 and pIJ773 used in this study.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Funding

This work was supported by Grants from the National High Technology Research and Development Program of China (2012AA092104), the National Natural Science Foundation of China (31570032 & 31171201), and the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China
(2)
CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences

References

  1. Fenical W, Jensen PR. Developing a new resource for drug discovery: marine actinomycete bacteria. Nat Chem Biol. 2006;2:666–73.View ArticleGoogle Scholar
  2. Jensen PR, Chavarria KL, Fenical W, Moore BS, Ziemert N. Challenges and triumphs to genomics-based natural product discovery. J Ind Microbiol Biot. 2014;41:203–9.View ArticleGoogle Scholar
  3. Bachmann BO, Van Lanen SG, Baltz RH. Microbial genome mining for accelerated natural products discovery: is a renaissance in the making? J Ind Microbiol Biot. 2014;41:175–84.View ArticleGoogle Scholar
  4. Hopwood D. Small things considered: the tip of the iceberg. 2008.Google Scholar
  5. Liu G, Chater KF, Chandra G, Niu G, Tan H. Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiol Mol Biol Rev. 2013;77:112–43.View ArticleGoogle Scholar
  6. Rutledge PJ, Challis GL. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nature Rev Microbiol. 2015;13:509–23.View ArticleGoogle Scholar
  7. Romero-Rodríguez A, Robledo-Casados I, Sánchez S. An overview on transcriptional regulators in Streptomyces. BBA-Gene Regulatory Mechanisms. 2015;1849:1017–39.Google Scholar
  8. Fowler-Goldsworthy K, Gust B, Mouz S, Chandra G, Findlay KC, Chater KF. The actinobacteria-specific gene wblA controls major developmental transitions in Streptomyces coelicolor A3 (2). Microbiology. 2011;157:1312–28.View ArticleGoogle Scholar
  9. Zheng F, Long Q, Xie J. The function and regulatory network of WhiB and WhiB-like protein from comparative genomics and systems biology perspectives. Cell Biochem Biophys. 2012;63:103–8.View ArticleGoogle Scholar
  10. Soliveri J, Gomez J, Bishai W, Chater K. Multiple paralogous genes related to the Streptomyces coelicolor developmental regulatory gene whiB are present in Streptomyces and other actinomycetes. Microbiology. 2000;146:333–43.View ArticleGoogle Scholar
  11. Nah JH, Park SH, Yoon HM, Choi SS, Lee CH, Kim ES. Identification and characterization of wblA-dependent tmcT regulation during tautomycetin biosynthesis in Streptomyces sp. CK4412. Biotechnol Adv. 2012;30:202–9.View ArticleGoogle Scholar
  12. Noh JH, Kim SH, Lee HN, Lee SY, Kim ES. Isolation and genetic manipulation of the antibiotic down-regulatory gene, wblA ortholog for doxorubicin-producing Streptomyces strain improvement. Appl Microbiol and Biot. 2010;86:1145–53.View ArticleGoogle Scholar
  13. Yu P, Liu SP, Bu QT, Zhou ZX, Zhu ZH, Huang FL, Li YQ. WblAch, a pivotal activator of natamycin biosynthesis and morphological differentiation in Streptomyces chattanoogensis L10, is positively regulated by AdpAch. Appl Environ Microbiol. 2014;80:6879–87.View ArticleGoogle Scholar
  14. Lu C, Liao G, Zhang J, Tan H. Identification of novel tylosin analogues generated by a wblA disruption mutant of Streptomyces ansochromogenes. Microb Cell Fact. 2015;14:1.View ArticleGoogle Scholar
  15. Zhang J, Jiang Y, Cao Y, Liu J, Zheng D, Chen X, Han L, Jiang C, Huang X. Violapyrones A-G, α-pyrone derivatives from Streptomyces violascens isolated from Hylobates hoolock feces. J Nat Prod. 2013;76:2126–30.View ArticleGoogle Scholar
  16. Tang X, Eitel K, Kaysser L, Kulik A, Grond S, Gust B. A two-step sulfation in antibiotic biosynthesis requires a type III polyketide synthase. Nat Chem Biol. 2013;9:610–5.View ArticleGoogle Scholar
  17. Nakano C, Ozawa H, Akanuma G, Funa N, Horinouchi S. Biosynthesis of aliphatic polyketides by type III polyketide synthase and methyltransferase in Bacillus subtilis. J Bacteriol. 2009;191:4916–23.View ArticleGoogle Scholar
  18. Song L, Barona-Gomez F, Corre C, Xiang L, Udwary DW, Austin MB, Noel JP, Moore BS, Challis GL. Type III polyketide synthase β-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining. J Am Chem Soc. 2006;128:14754–5.View ArticleGoogle Scholar
  19. Aizawa T, Kim SY, Takahashi S, Koshita M, Tani M, Futamura Y, Osada H, Funa N. Alkyldihydropyrones, new polyketides synthesized by a type III polyketide synthase from Streptomyces reveromyceticus. J Antibiot (Tokyo). 2014;67(12):819–23.View ArticleGoogle Scholar
  20. Funa N, Ozawa H, Hirata A, Horinouchi S. Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii. Proc Natl Acad Sci USA. 2006;103:6356–61.View ArticleGoogle Scholar
  21. Katsuyama Y, Ohnishi Y. Type III polyketide synthases in microorganisms. Methods Enzymol. 2012;515:359–77.View ArticleGoogle Scholar
  22. Morita H, Yamashita M, Shi SP, Wakimoto T, Kondo S, Kato R, Sugio S, Kohno T, Abe I. Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase. Proc Natl Acad Sci USA. 2011;108:13504–9.View ArticleGoogle Scholar
  23. Chemler JA, Buchholz TJ, Geders TW, Akey DL, Rath CM, Chlipala GE, Smith JL, Sherman DH. Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor. J Am Chem Soc. 2012;134:7359–66.View ArticleGoogle Scholar
  24. Kang SH, Huang J, Lee HN, Hur YA, Cohen SN, Kim ES. Interspecies DNA microarray analysis identifies WblA as a pleiotropic down-regulator of antibiotic biosynthesis in Streptomyces. J Bacteriol. 2007;189:4315–9.View ArticleGoogle Scholar
  25. Rabyk M, Ostash B, Rebets Y, Walker S, Fedorenko V. Streptomyces ghanaensis pleiotropic regulatory gene wblA gh influences morphogenesis and moenomycin production. Biotechnol Lett. 2011;33:2481–6.View ArticleGoogle Scholar
  26. Shin HJ, Lee HS, Lee JS, Shin J, Lee MA, Lee HS, Lee YJ, Yun J, Kang JS. Violapyrones H and I, new cytotoxic compounds isolated from Streptomyces sp. associated with the marine starfish Acanthaster planci. Mar Drugs. 2014;12:3283–91.View ArticleGoogle Scholar
  27. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1982.Google Scholar
  28. Paget MS, Chamberlin L, Atrih A, Foster SJ, Buttner MJ. Evidence that the extracytoplasmic function sigma factor σE is required for normal cell wall structure in Streptomyces coelicolor A3 (2). J Bacteriol. 1999;181:204–11.Google Scholar
  29. Gust B, Challis GL, Fowler K, Kieser T, Chater KF. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA. 2003;100:1541–6.View ArticleGoogle Scholar
  30. Zhang Y, Huang H, Xu S, Wang B, Ju J, Tan H, Li W. Activation and enhancement of Fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology. Microb Cell Fact. 2015;14:64.View ArticleGoogle Scholar
  31. Weber T, Blin K, Duddela S, Krug D, Kim H, Bruccoleri R, Lee S, Fischbach M, Muller R, Wohlleben W, Breitling R, Takano E, Medema M. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015;43(1):W237–43.View ArticleGoogle Scholar
  32. Ishikawa J, Hotta K. FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. FEMS Microbiol Lett. 1999;174:251–3.View ArticleGoogle Scholar
  33. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticleGoogle Scholar
  34. Gust B, Chandra G, Jakimowicz D, Yuqing T, Bruton CJ, Chater KF. Lambda red-mediated genetic manipulation of antibiotic-producing Streptomyces. Adv Appl Microbiol. 2004;54:107–28.View ArticleGoogle Scholar
  35. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces genetics. Norwich: John Innes Foundation; 2000.Google Scholar

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© The Author(s) 2016

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