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Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces.

ABSTRACT: 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 P_ecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal P_ecf17 promoter was determined, and a P_ecf17 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.

SUBMITTER: Zhao X

PROVIDER: S-EPMC9194655 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The SCO4117 ECF Sigma Factor Pleiotropically Controls Secondary Metabolism and Morphogenesis in Streptomyces coelicolor.

Project description:Extracytoplasmic function (ECF) sigma factors are a major type of bacterial signal-transducers whose biological functions remain poorly characterized in streptomycetes. In this work we studied SCO4117, a conserved ECF sigma factor from the ECF52 family overexpressed during substrate and aerial mycelium stages. The ECF52 sigma factors harbor, in addition to the ECF sigma factor domain, a zinc finger domain, a transmembrane region, a proline-rich C-terminal extension, and a carbohydrate-binding domain. This class of ECF sigma factors is exclusive to Actinobacteria. We demonstrate that SCO4117 is an activator of secondary metabolism, aerial mycelium differentiation, and sporulation, in all the culture media (sucrose-free R5A, GYM, MM, and SFM) analyzed. Aerial mycelium formation and sporulation are delayed in a SCO4117 knockout strain. Actinorhodin production is delayed and calcium-dependent antibiotic production is diminished, in the ?SCO4117 mutant. By contast, undecylprodigiosin production do not show significant variations. The expression of genes encoding secondary metabolism pathways (deoxysugar synthases, actinorhodin biosynthetic genes) and genes involved in differentiation (rdl, chp, nepA, ssgB) was dramatically reduced (up to 300-fold) in the SCO4117 knockout. A putative motif bound, with the consensus "CSGYN-17bps-SRHA" sequence, was identified in the promoter region of 29 genes showing affected transcription in the SCO4117 mutant, including one of the SCO4117 promoters. SCO4117 is a conserved gene with complex regulation at the transcriptional and post-translational levels and the first member of the ECF52 family characterized.

| S-EPMC5826349 | biostudies-literature

Streptomyces griseus transcriptome analysis in delta sigD, encoding an ECF-sigma factor

2011-02-23 | GSE27426 | GEO

SCO4117 ECF sigma factor pleiotropically controls secondary metabolism and morphogenesis in Streptomyces coelicolor

Project description:Extracytoplasmic function (ECF) sigma factors are a major type of bacterial signal transducer whose biological functions remain poorly characterised in streptomycetes. In this work, we studied SCO4117, a conserved ECF sigma factor from the ECF52 family, a class of ECF sigma factors exclusive to Actinobacteria. We demonstrate that SCO4117 is a pleiotropic activator of secondary metabolism, germination, aerial mycelium differentiation and sporulation. The expression of genes encoding secondary metabolism pathways (deoxysugar synthases, actinorhodin biosynthetic genes), and genes involved in differentiation (bld, whi, rdl, chp, nepA, ssgB, scbR), was dramatically reduced (up to 300-fold) in a SCO4117 knockout strain. SCO4117 expression depends on its position in the chromosome, as the knockout mutant phenotype can only be fully complemented when the SCO4117 ORF is restored in the chromosome. A strain expressing a truncated version of SCO4117 harbouring only the ECF sigma factor domain, has an opposite phenotype to that observed in the knockout strain, consisting of overproduction of antibiotics (actinorhodin, CDA, cryptic antibiotics) and acceleration of differentiation (aerial mycelium differentiation and sporulation), suggesting a complex interaction between the SCO4117 domains that affects protein activity. Some genes from predicted secondary metabolite clusters (cryptic pathways) were up-regulated compared to the wild strain, indicating that this truncated version of the SCO4117 protein might be useful to activate cryptic pathways. Overall, in this work we demonstrate the pleiotropic effects on the regulation of secondary metabolism and differentiation of SCO4117, the first member of the ECF52 family to be characterised. SCO4117 is a conserved gene overexpressed during the substrate and aerial mycelium stages, showing complex regulation at the transcriptional and post-translational levels.

2018-03-07 | GSE107661 | GEO

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR.

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Streptomyces griseus transcriptome analysis in delta sigD, encoding an ECF-sigma factor

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2011-02-23 | E-GEOD-27426 | biostudies-arrayexpress

Sigma factor WhiGch positively regulates natamycin production in Streptomyces chattanoogensis L10

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2015-07-02 | GSE58995 | GEO

A Novel extracytoplasmic function (ECF) sigma factor regulates virulence in Pseudomonas aeruginosa.

Project description:Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a beta-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen.

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Mycobacterium tuberculosis requires the ECF sigma factor SigE to arrest phagosome maturation.

Project description:SigE represents one of the best characterized alternative sigma factors of Mycobacterium tuberculosis, playing a major role in the response to several environmental stresses and essential for growth in macrophages and virulence. In previous work we demonstrated that a mutant of M. tuberculosis in which the sigE gene was disrupted by a cassette conferring hygromycin resistance is a promising vaccine candidate conferring better protection than Mycobacterium bovis BCG in a mouse model of infection. In this work we describe the construction of a new unmarked mutant in which the entire sigE gene was disrupted in order to fulfill the requirements of the Geneva consensus to enter clinical trials. After showing that the phenotype of this mutant is superimposable to that of the previous one, we further characterized the role of SigE in the M tuberculosis intracellular behavior showing that it is dispensable for replication in human pneumocytes, while it is essential for the arrest of phagosome maturation in THP-1-derived macrophages.

| S-EPMC4182583 | biostudies-literature

Characterization and in vivo regulon determination of an ECF sigma factor and its cognate anti-sigma factor in Nostoc punctiforme.

Project description:Based on primary sequence comparisons and genomic context, Npun_F4153 (SigG)/Npun_F4154 (SapG) of the cyanobacterium Nostoc punctiforme were hypothesized to encode an ECF sigma factor/anti-sigma factor pair. Transcription of sigG increased in heterocysts and akinetes, and after EDTA treatment. Interaction between SigG and the predicted cytoplasmic domain of SapG was observed in vitro. A SigG-GFP translational fusion protein localized to the periphery of vegetative cells in vivo, but lost this association following heat stress. A sigG mutant was unable to survive envelope damage caused by heat or EDTA, but was able to form functional heterocysts. Akinetes in the mutant strain appeared normal, but these cultures were less resistant to lysozyme and cold treatments than those of the wild-type strain. The SigG in vivo regulon was determined before and during akinete differentiation using DNA microarray analysis, and found to include multiple genes with putative association to the cell envelope. Mapped promoters common to both arrays enabled identification of a SigG promoter-binding motif that was supported in vivo by reporter studies, and in vitro by run-off transcription experiments. These findings support SigG/SapG as a sigma/anti-sigma pair involved in repair of envelope damage resulting from exogenous sources or cellular differentiation.

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Identification and characterization of the cognate anti-sigma factor and specific promoter elements of a T. tengcongensis ECF sigma factor.

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