Modulation of Multiple Gene Clusters' Expression by the PAS-LuxR Transcriptional Regulator PteF.

PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from . Its mutation drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator.

Understanding microRNAs in the Context of Infection to Find New Treatments against Human Bacterial Pathogens.

The development of RNA-based anti-infectives has gained interest with the successful application of mRNA-based vaccines. Small RNAs are molecules of RNA of <200 nucleotides in length that may control the expression of specific genes. Small RNAs include small interference RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), or microRNAs (miRNAs).

Production, Detection, Extraction, and Quantification of Polyene Antibiotics.

Polyene antibiotics are macrolide antifungal compounds obtained by fermentation of producer Streptomyces strains. Here we describe commonly used methods for polyene production, detection, and their subsequent extraction and purification. While bioassays are used to detect these compounds based on their biological activity, quantification by spectrophotometry or high-performance liquid chromatography (HPLC ) relies on their physiochemical properties and is more reliable.

Structural and Bioactivity Characterization of Filipin Derivatives from Engineered Strains Reveals Clues for Reduced Haemolytic Action.

The rise in the number of immunocompromised patients has led to an increased incidence of fungal infections, with high rates of morbidity and mortality. Furthermore, misuse of antifungals has boosted the number of resistant strains to these agents; thus, there is urgent need for new drugs against these infections. Here, the in vitro antifungal activity of filipin III metabolic intermediates has been characterized against a battery of opportunistic pathogenic fungi-, , , , , , , , and -using the Clinical and Laboratory Standards Institute broth microdilution method.

The Streptomyces filipinensis Gamma-Butyrolactone System Reveals Novel Clues for Understanding the Control of Secondary Metabolism.

γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of , the producer of the antifungal agent filipin, has been investigated. Inactivation of (for γ-utyrolactone eceptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of , a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively.

Activation of Secondary Metabolite Gene Clusters in by the PimM Regulator of .

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple strains. In this work we show that constitutive expression of in ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism.

Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor.

The biosynthesis of the antifungal filipin in Streptomyces filipinensis is very sensitive to phosphate regulation. Concentrations as low as 2.5 mM block filipin production.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide.

Biotechnological production and application of the antibiotic pimaricin: biosynthesis and its regulation.

Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides.

Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives.

Background: Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield.

Pathway-specific regulation revisited: cross-regulation of multiple disparate gene clusters by PAS-LuxR transcriptional regulators.

PAS-LuxR regulators are highly conserved proteins devoted to the control of antifungal production by binding to operators located in given promoters of polyene biosynthetic genes. The canonical operator of PimM, archetype of this class of regulators, has been used here to search for putative targets of orthologous protein PteF in the genome of Streptomyces avermitilis, finding 97 putative operators outside the pentaene filipin gene cluster (pte). The processes putatively affected included genetic information processing; energy, carbohydrate, and lipid metabolism; DNA replication and repair; morphological differentiation; secondary metabolite biosynthesis; and transcriptional regulation, among others.

PAS-LuxR transcriptional control of filipin biosynthesis in S. avermitilis.

The DNA region encoding the filipin gene cluster in Streptomyces avermitilis (pte) contains a PAS-LuxR regulatory gene, pteF, orthologue to pimM, the final pathway-specific positive regulatory protein of pimaricin biosynthesis in Streptomyces natalensis. Gene replacement of the gene from S. avermitilis chromosome resulted in a severe loss of filipin production and delayed spore formation in comparison to that of the wild-type strain, suggesting that it acts as a positive regulator of filipin biosynthesis and that it may also have a role in sporulation.

Genome-wide analysis of the regulation of pimaricin production in Streptomyces natalensis by reactive oxygen species.

To investigate the molecular mechanisms that interplay between oxygen metabolism and secondary metabolism in Streptomyces natalensis, we compared the transcriptomes of the strains CAM.02 (ΔsodF), pimaricin under-producer phenotype, and CAM.04 (ΔahpCD), pimaricin over-producer phenotype, with that of the wild type at late exponential and stationary growth phases.

Draft genome of Streptomyces tsukubaensis NRRL 18488, the producer of the clinically important immunosuppressant tacrolimus (FK506).

The macrocyclic polyketide tacrolimus (FK506) is a potent immunosuppressant that prevents T-cell proliferation produced solely by Streptomyces species. We report here the first draft genome sequence of a true FK506 producer, Streptomyces tsukubaensis NRRL 18488, the first tacrolimus-producing strain that was isolated and that contains the full tacrolimus biosynthesis gene cluster.

Hierarchical control on polyene macrolide biosynthesis: PimR modulates pimaricin production via the PAS-LuxR transcriptional activator PimM.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimR(SARP)) was expressed in Escherichia coli as a glutathione S-transferase (GST)-fused protein.

LAL regulators SCO0877 and SCO7173 as pleiotropic modulators of phosphate starvation response and actinorhodin biosynthesis in Streptomyces coelicolor.

LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S.

Functional conservation of PAS-LuxR transcriptional regulators in polyene macrolide biosynthesis.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the PAS-LuxR transcriptional activator PimM. Expression of target genes in this strain is positively regulated by binding of the regulator to 14-nucleotide sites showing dyad symmetry, and overlapping the -35 element of each promoter. These sequences have been found in the upstream regions of genes belonging to different polyene biosynthetic gene clusters.

Molecular control of polyene macrolide biosynthesis: direct binding of the regulator PimM to eight promoters of pimaricin genes and identification of binding boxes.

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimM(ΔPAS)), and forms containing just the DNA-binding domain (DBD) (PimM(DBD)) were overexpressed in Escherichia coli as GST-fused proteins.

Structure of cytochrome P450 PimD suggests epoxidation of the polyene macrolide pimaricin occurs via a hydroperoxoferric intermediate.

We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with improved pharmacologic properties.

PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis.

Background: Polyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer Streptomyces natalensis, which elicits polyene production in a manner characteristic of quorum sensing.

Enzymology of the polyenes pimaricin and candicidin biosynthesis.

Pimaricin and candicidin are prototypical representatives of the "small" and the "aromatic" polyene macrolides, respectively. Pimaricin, produced by Streptomyces natalensis, is an important antifungal agent used in human therapy for the treatment of fungal keratitis, and in the food industry to prevent mould contamination. Five large polyketide synthase subunits are implicated in the formation of the pimaricin macrolactone ring, while P450 mono-oxygenases and a glycosyltransferase are responsible for ring "decoration.

Microbial cholesterol oxidases: bioconversion enzymes or signal proteins?

Cholesterol oxidases (3beta-hydroxysterol oxidases; EC 1.1.3.

Biosynthetic engineering of polyene macrolides towards generation of improved antifungal and antiparasitic agents.

Polyene macrolides are potent antifungal agents that are also active against parasites, enveloped viruses and prion diseases. They are medically important as antifungal antibiotics but their therapeutic use is limited by serious side effects. In recent years there has been considerable progress in genetic analysis and manipulation of the streptomycetes that produce nystatin, amphotericin B, candicidin, pimaricin and rimocidin/CE-108-related polyenes.

PimM, a PAS domain positive regulator of pimaricin biosynthesis in Streptomyces natalensis.

Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 579 bp gene, pimM, whose deduced product (192 aa) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimM combines an N-terminal PAS domain with a C-terminal helix-turn-helix (HTH) motif of the LuxR type. Gene replacement of pimM from the Streptomyces natalensis chromosome with a mutant version lacking the HTH DNA-binding domain resulted in complete loss of pimaricin production, suggesting that PimM is a positive regulator of pimaricin biosynthesis.

Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin.

The gene cluster responsible for pimaricin biosynthesis in Streptomyces natalensis contains a cholesterol oxidase-encoding gene (pimE) surrounded by genes involved in pimaricin production. Gene-inactivation and -complementation experiments revealed that pimE encodes a functional cholesterol oxidase and, surprisingly, that it is also involved in pimaricin biosynthesis. This extracellular enzyme was purified from S.