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

<p>Abstract</p> <p>Background</p> <p>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 <it>Streptomyces natalensis</it>, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from <it>S. natalensis </it>in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.</p> <p>Results</p> <p>Adjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.</p> <p>Conclusion</p> <p>This report describes the involvement of an amino acid exporter (encoded by <it>pimT </it>in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.</p

Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis

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 Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex

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. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism

Modulación de la síntesis de macrólidos poliénicos: el paradigma de la pimaricina = Modulation of polyene macrolides synthesis: pimaricin as a paradigm

302 p.Los policétidos constituyen una vasta família de metabolitos secundarios entre los que se cuentan moléculas de gran importancia clínica y económica, como antibacterianos, antitumorales, anticolesterolémicos, inmunosupresores o antitumorales. Entre ellos se cuentan además los macrólidos poliénicos, compuestos producidos por actinobacterias del género Streptomyces y que presentan una elevada actividad antifúngica que convierte a algunos de ellos en la principal alternativa terapeútica frente a las cada vez más prevalentes micosis opotunistas invasivas y otras infecciones causadas por hongos. La pimaricina, un polieno producido por S. natalensis con aplicaciones clínicas y en la industria alimentaria, constituye un interesante paradigma para ayudarnos a comprender los mecanismos biosintéticos y regulatorios que gobiernan la producción del conjunto de los macrólidos poliénicos. La comprensión de la regulación de la producción los metabolitos secundarios proporciona algunas de las vías más intuitivas y fácilmente manipulables cuando lo que se busca es el aumento su producción industrial, pero hasta la fecha se sabe muy poco acerca del funcionamiento de estos mecanismos en el caso de los polieno

Beyond Soil-Dwelling Actinobacteria: Fantastic Antibiotics and Where to Find Them

Bacterial secondary metabolites represent an invaluable source of bioactive molecules for the pharmaceutical and agrochemical industries. Although screening campaigns for the discovery of new compounds have traditionally been strongly biased towards the study of soil-dwelling Actinobacteria, the current antibiotic resistance and discovery crisis has brought a considerable amount of attention to the study of previously neglected bacterial sources of secondary metabolites. The development and application of new screening, sequencing, genetic manipulation, cultivation and bioinformatic techniques have revealed several other groups of bacteria as producers of striking chemical novelty. Biosynthetic machineries evolved from independent taxonomic origins and under completely different ecological requirements and selective pressures are responsible for these structural innovations. In this review, we summarize the most important discoveries related to secondary metabolites from alternative bacterial sources, trying to provide the reader with a broad perspective on how technical novelties have facilitated the access to the bacterial metabolic dark matter

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

International audienceThe biosynthesis of small-size polyene macrolides is ultimately con-trolled by acouple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxRfamily (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encodinggene and activates its transcription, and in turn, the gene product of the latter acti-vates transcription from various promoters of the polyene gene cluster directly. Theprimary operator of PimR, the archetype of SARP-LAL regulators, contains three hep-tameric directrepeats separated by four-nucleotide spacers, but the regulator canalso bind a secondary operator with only two direct repeats separated by a3-nucleotide spacer, both located in the promoter region of its unique target gene,pimM. A similar arrangement of operators has been identified for PimR counterpartsencoded by gene clusters for different antifungal secondary metabolites, includingnot only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cyclohex-imide. Here, we used promoter engineering and quantitative transcriptional analysesto determine the contributions of the different heptameric repeats to transcriptionalactivation and final polyene production. Optimized promoters have thus been devel-oped. Deletion studies and electrophoretic mobility assays were used for the defini-tion of DNA-binding boxes formed by 22-nucleotide sequences comprising twoconserved heptameric direct repeats separated by four-nucleotide less conservedspacers. The cooperative binding of PimRSARPappears to be the mechanism involvedin the binding of regulator monomers to operators, and at least two protein mono-mers are required for efficient binding

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

International audiencePAS-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 putativelyaffected included genetic information processing; energy, carbohydrate, and lipid metabolism; DNA replication and repair; morphological differentiation; secondary metabolite biosynthesis; and transcriptional regulation, among others.Seventeen of these operators were selected, and their binding to PimM DNA-binding domain was assessed by electrophoretic mobility shift assays. Strikingly, the protein bound all predicted operators suggesting a direct control over targeted processes. As a proof of concept, we studied the biosynthesis of the ATP-synthase inhibitor oligomycin whose gene cluster included two operators. Regulator mutants showed a severe loss of oligomycin production, whereas gene complementation of the mutant restored phenotype, and gene duplication in the wild-type strain boosted oligomycin production. Comparative gene expression analyses in parental and mutant strains by reverse transcription-quantitative polymerase chain reaction of selected olm genes corroborated production results. These results demonstrate that PteF is able to crossregulate the biosynthesis of two related secondary metabolites, filipin and oligomycin, but might be extended to all the processes indicated above. This study highlights the complexity of the network of interactions in which PAS-LuxR regulators are involved and opens new possibilities for the manipulation of metabolite production in Streptomycetes

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

International audienceThe 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. Complementation of the mutant with a single copy of the gene integrated into the chromosome restored wild-type phenotypes. Heterologous complementation with the regulatory counterpart from S. natalensis also restored parental phenotypes. Gene expression analyses in S. avermitilis wild-type and the mutant by reverse transcription-quantitative polymerase chain reaction of the filipin gene cluster suggested the targets for the regulatory protein. Transcription start points of all the genes of the cluster were studied by 5′-rapid amplification of complementary DNA ends. Transcription start point analysis of the pteF gene revealed that the annotated sequence in the databases is incorrect. Confirmation of target promoters was performed by in silico search of binding sites among identified promoters and the binding of the orthologous regulator for pimaricin biosynthesis PimM to gene promoters by electrophoretic mobility shift assays. Precise binding regions were investigated by DNAse I protection studies. Our results indicate that PteF activates the transcription from two promoters of polyketide synthase genes directly, and indirectly of other genes of the cluster