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Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor ? (AntA.).

ABSTRACT: Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF) RNA polymerase sigma factor named ? (AntA) which represents a new sub-family of ECF ? factors that is only found in antimycin producing strains. We show that ? (AntA) controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. ? (AntA) is highly conserved in antimycin producing strains and the -10 and -35 elements at the ? (AntA) regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all ? (AntA) proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.

SUBMITTER: Seipke RF

PROVIDER: S-EPMC3933326 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σ (AntA.).

Seipke Ryan F RF Patrick Elaine E Hutchings Matthew I MI

PeerJ 20140206

Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant) gen ...[more]

PMID: 24688837

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Project description:Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a few them are expressed in the laboratory; thus, poor understanding of how natural-product biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species, and their regulation is atypical compared to that of most other natural products. Here we demonstrate that antimycin production by Streptomyces albus S4 is regulated by FscRI, a PAS-LuxR family cluster-situated regulator of the polyene antifungal agent candicidin. We report that heterologous production of antimycins by Streptomyces coelicolor is dependent on FscRI and show that FscRI activates the transcription of key biosynthetic genes. We also demonstrate through chromatin immunoprecipitation sequencing that FscRI regulation is direct, and we provide evidence that this regulation strategy is conserved and unique to short-form antimycin gene clusters. Our study provides direct in vivo evidence of the cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products and expands the paradigmatic understanding of the regulation of secondary metabolism. IMPORTANCE Natural products produced by members of the phylum Actinobacteria underpin many industrially and medically important compounds; however, the majority of the ~30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. Understanding the diversity of regulatory strategies controlling the expression of these pathways is therefore critical if their biosynthetic potential is to be explored for new drug leads. Our findings reveal that the candicidin cluster-situated regulator FscRI coordinately controls the biosynthesis of both candicidin and antimycin, which is the first observation of cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products. We anticipate that this will emerge as a major strategy by which members of the phylum Actinobacteria coordinately produce natural products, which will advance our understanding of how the expression of secondary metabolism is controlled and will aid the pursuit of "silent" biosynthetic pathway activation.

Project description:The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

Project description:BackgroundThe expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response.ResultsTranscription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed.ConclusionsThe rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.

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Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor ? (AntA.).

Publications

Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σ (AntA.).

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