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In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24.

ABSTRACT: Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis.

SUBMITTER: Yan L

PROVIDER: S-EPMC5373594 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24.

Yan Lan L Zhang Qizhong Q Virolle Marie-Joelle MJ Xu Delin D

PloS one 20170330 3

Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigmen ...[more]

PMID: 28358920

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Project description:Streptomyces lividans TK24 is a relevant Gram-positive soil inhabiting bacterium and one of the model organisms of the genus Streptomyces. It is known for its potential to produce secondary metabolites, antibiotics, and other industrially relevant products. S. lividans TK24 is the plasmid-free derivative of S. lividans 66 and a close genetic relative of the strain Streptomyces coelicolor A3(2). In this study, we used transcriptome and proteome data to improve the annotation of the S. lividans TK24 genome. The RNA-seq data of primary 5'-ends of transcripts were used to determine transcription start sites (TSS) in the genome. We identified 5,424 TSS, of which 4,664 were assigned to annotated CDS and ncRNAs, 687 to antisense transcripts distributed between 606 CDS and their UTRs, 67 to tRNAs, and 108 to novel transcripts and CDS. Using the TSS data, the promoter regions and their motifs were analyzed in detail, revealing a conserved -10 (TAnnnT) and a weakly conserved -35 region (nTGACn). The analysis of the 5' untranslated region (UTRs) of S. lividans TK24 revealed 17% leaderless transcripts. Several cis-regulatory elements, like riboswitches or attenuator structures could be detected in the 5'-UTRs. The S. lividans TK24 transcriptome contains at least 929 operons. The genome harbors 27 secondary metabolite gene clusters of which 26 could be shown to be transcribed under at least one of the applied conditions. Comparison of the reannotated genome with that of the strain Streptomyces coelicolor A3(2) revealed a high degree of similarity. This study presents an extensive reannotation of the S. lividans TK24 genome based on transcriptome and proteome analyses. The analysis of TSS data revealed insights into the promoter structure, 5'-UTRs, cis-regulatory elements, attenuator structures and novel transcripts, like small RNAs. Finally, the repertoire of secondary metabolite gene clusters was examined. These data provide a basis for future studies regarding gene characterization, transcriptional regulatory networks, and usage as a secondary metabolite producing strain.

Project description:The comprehensive transcriptome of Streptomyces lividans TK24 in a biolector milliliter-scale cultivation system was compared in detail against an established 1000-fold larger lab-scale bioreactor system. For main-cultures, the modified minimal medium NMMP (D'Huys et al. 2011) was applied, containing 2.07 g/L NaH2PO4 x 1 H2O, 2.6 g/L K2HPO4, 0.6 MgSO4 x 7 H2O, 3.0 g/L (NH4)2SO4, 10 g/L D-glucose and 5 g/L BactoTM Casamino acids as well as several trace elements (1 mg/L ZnSO4 x 7 H2O, 1 mg/L FeSO4 x 7 H2O, 1 mg/L MnCl2 x 4 H2O, 1 mg/L CaCl2). pH was adjusted to 6.8 using 1 M KOH or 1 M H2SO4. In all MTP BioLector (BL) cultivations and in some bioreactor experiments (when indicated) 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) was added as biological inert buffer system. Except for the phosphate buffer, all other components were prepared as stock solutions and sterilized separately. For MTP based cultivations the BL device, all stock solutions kept frozen at -20 C in aliquots sufficient for 50 mL final medium, to ensure identical conditions between experimental runs. For the system comparison experiments the same medium charge was used for all cultivations. 1 mL MTP-based BL cultivations MTP cultivations were performed in 48 well FlowerPlates (m2p-labs GmbH, Baesweiler, Germany), covered by a gas-permeable sealing foil (m2p-labs GmbH, Baesweiler, Germany). 1000 L cultivation medium was inoculated to a final OD600 of 0.2. Temperature and humidity was controlled in the incubation chamber of the BL device at 30 C and 89 % respectively. All signals (BS, DO, and pH), were measured with a cycle time of around 10 min. Altogether, 96 identical parallel milliliter-scale cultivations were performed in two separate but identical BL devices. One of these devices was additionally embedded in a lab-robotic liquid handling unit in the style of (Unthan et al. 2015; Rohe et al. 2012), to provide automated hourly sampling for substrate and cell dry weight analysis. The cultivations in the second BL unit served to generate samples for the omics-based analysis, discussed in the later sections. Bioreactor cultivations were carried out in a 1.5 L lab-scale system (DasGip, Jlich, Germany), equipped with two six blade, 46 mm Rushton turbines as well as sensors for DO (Hamilton, Visiferm DO 225) and pH (MettlerToledo, 405-DPAS-SC-K8S/225/120). Temperature was controlled at 30 C; evaporation was minimized by offgas cooling at 10 C. 700 mL phosphate buffer was sterilized directly within the reactor glass vessel; all other media components were added sterile and premixed subsequently by using a 500 mL feeding flask. 0.5 mL anti-foam (AF204 (Sigma, MO, USA)) was added before DO calibration. The final 1000 mL medium was inoculated with washed mycelium to a final OD600 of 0.2. Both, aeration and agitation were set to fixed values at 0.5 vvm and 800 rpm respectively. For both techniques samples were taken during the late growth phase, two hours after the switch to process phase II.

Dataset Information

In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24.

Publications

In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24.

Similar Datasets

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