Sigma factor WhiGch positively regulates natamycin production in Streptomyces chattanoogensis L10
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ABSTRACT:
ORGANISM(S): Streptomyces chattanoogensis
PROVIDER: GSE58995 | GEO | 2015/07/02
SECONDARY ACCESSION(S): PRJNA254066
REPOSITORIES: GEO
Similar Datasets
Project description:In LG01 there were a total of 861 genes displaying at least a two-fold change (P<0.05), including 470 genes with lower and 391 genes with higher expressions In this study, RNA isolated from LG01 or wild type Streptomyces chattanoogensis L10, was used to acquire expression profiles of a total of 8,117 protein-coding genes in S. chattanoogensis L10, leading to the successful construction of different expression profiles between whiGch deletion mutant and wild type S. chattanoogensis L10.
2015-07-02 | E-GEOD-58995 | biostudies-arrayexpress
Project description:scnA (GenBank: ADX66464.1) and scnB (GenBank: ADX66465.1) are ABC transporters located in the biosysnthesis gene cluster of natamycin in Streptomyces chattanoogensis. The two genes are presumbly involved in natamycin efflux. Our goal of this expriment was to investigate the effect of scnA and scnB inactivation on the gene expression in the whole genome. In ∆scnAB there were a total of 219 genes displaying at least a two-fold change (P<0.05), including 89 genes with lower and 130 genes with higher expressions
2015-09-17 | GSE73086 | GEO
Project description:scnA (GenBank: ADX66464.1) and scnB (GenBank: ADX66465.1) are ABC transporters located in the biosysnthesis gene cluster of natamycin in Streptomyces chattanoogensis. The two genes are presumbly involved in natamycin efflux. Our goal of this expriment was to investigate the effect of scnA and scnB inactivation on the gene expression in the whole genome. In ∆scnAB there were a total of 219 genes displaying at least a two-fold change (P<0.05), including 89 genes with lower and 130 genes with higher expressions In this study, RNA isolated from ∆scnAB or wild type Streptomyces chattanoogensis L10, was used to acquire expression profiles of a total of 8,117 protein-coding genes in S. chattanoogensis L10, leading to the successful construction of different expression profiles between scnA and scnB deletion mutant and wild type S. chattanoogensis L10.
2015-09-17 | E-GEOD-73086 | biostudies-arrayexpress
Project description:Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer, Streptomyces chattanoogensis L10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT in S. chattanoogensis L10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.
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Project description:Detailed mechanisms of WhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, a Streptomyces chattanoogensis L10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression of wblAch was shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAch has specific DNA-binding activity for the promoter region of wblAch. Gene disruption and genetic complementation revealed that WblAch acts in a positive manner to regulate natamycin production. When wblAch was overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels of whi genes (including whiA, whiB, whiH, and whiI) were severely depressed in the ΔwblAch mutant, suggesting that WblAch plays a part in morphological differentiation by influencing the expression of the whi genes.
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Project description:Alternative σ factors in bacteria redirect RNA polymerase to recognize alternative promoters, thereby facilitating coordinated gene expression necessary for adaptive responses. The gene sig8 (sav_741) in Streptomyces avermitilis encodes an alternative σ factor, σ8, highly homologous to σB in Streptomyces coelicolor. Studies reported here demonstrate that σ8 is an important regulator of both avermectin production and stress responses in S. avermitilis. σ8 inhibited avermectin production by indirectly repressing expression of cluster-situated activator gene aveR, and by directly initiating transcription of its downstream gene sav_742, which encodes a direct repressor of ave structural genes. σ8 had no effect on cell growth or morphological differentiation under normal growth conditions. Growth of a sig8-deletion mutant was less than that of wild-type strain on YMS plates following treatment with heat, H2O2, diamide, NaCl, or KCl. sig8 transcription was strongly induced by these environmental stresses, indicating response by σ8 itself. A series of σ8-dependent genes responsive to heat, oxidative and osmotic stress were identified by EMSAs, qRT-PCR and in vitro transcription experiments. These findings indicate that σ8 plays an important role in mediating protective responses to various stress conditions by activating transcription of its target genes. Six σ8-binding promoter sequences were determined and consensus binding sequence BGVNVH-N15-GSNNHH (B: C, T or G, V: A, C or G, S: C or G, H: A, C or T, N: any nucleotide) was identified, leading to prediction of the σ8 regulon. The list consists of 940 putative σ8 target genes, assignable to 17 functional groups, suggesting the wide range of cellular functions controlled by σ8 in S. avermitilis.
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Project description:The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3 μ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 μg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.
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Project description:Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase ?-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.
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Project description:Sigma factors and sigma factor-related mechanisms control antibiotic production in Streptomyces. In this contribution, the orf21 gene was overexpressed in the wild-type strain of Streptomyces clavuligerus ATCC2764, yielding S. clavuligerus/pIORF21, to further evaluate its regulatory effect on clavulanic acid (CA) biosynthesis under different culture medium conditions. The orf21 overexpression, regulated under the constitutive promoter ermE*, led to 2.6-fold increase in CA production in GSPG medium, and a 1.8-fold decrease using ISP medium. As for GYM and MYM media, S. clavuligerus/pIORF21 strain showed higher aerial mycelium production compared to control. Glycerol uptake rate profile was affected by orf21 overexpression. Furthermore, in GSPG, S. clavuligerus/pIORF21 slightly increased the expression of adpA and gcas genes, whilst, in ISP, the claR gene expression was drastically reduced, which is consistent with a decreased CA production, observed in this medium. These findings suggest the protein encoded by the orf21 gene plays a role in the regulation of CA biosynthesis as a response to the nutritional composition of the medium.
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Project description:Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ΔdepR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor.
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