ABSTRACT
The γ-butyrolactone autoregulator signaling cascade is widely distributed among Streptomyces species as an important regulatory system of secondary metabolism. In Streptomyces lavendulae FRI-5, a γ-butyrolactone autoregulator IM-2 and the IM-2 specific receptor FarA control production of the blue pigment indigoidine together with two types of antibiotics: d-cycloserine and the nucleoside antibiotics. Here, we demonstrated by in silico analysis that farR2 (a farA homologue), which is located in a cluster of regulatory genes including farA, belongs to the family of pseudoreceptor regulator genes, and that the expression of farR2 is controlled by the IM-2/FarA regulatory system. Disruption of farR2 resulted in delayed production of indigoidine and in transcriptional derepression of the clustered far regulatory genes. Moreover, FarR2 bound to the FarA-binding sequences in the promoter regions of the regulatory genes that were downregulated by FarR2.
Subject(s)
Piperidones/metabolism , Receptors, GABA-A/metabolism , Streptomyces/metabolism , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Computer Simulation , Down-Regulation , Gene Expression Regulation, Bacterial , Genes, Regulator/genetics , Multigene Family/genetics , Promoter Regions, Genetic/genetics , Receptors, GABA-A/genetics , Secondary Metabolism , Streptomyces/genetics , Transcription, Genetic/geneticsABSTRACT
The Streptomyces antibiotic regulatory protein (SARP) family regulators have been shown to control the production of secondary metabolites in many Streptomyces species as the most downstream regulators in the regulatory cascade. Streptomyces lavendulae FRI-5 produces a blue pigment (indigoidine) together with two types of antibiotics: D-cycloserine and the nucleoside antibiotics. The production of these secondary metabolites is governed by a signaling system consisting of a γ-butyrolactone, IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl-γ-butanolide], and its cognate receptor, FarA. Here, we characterized two regulatory genes of the SARP family, farR3 and farR4, which are tandemly located in the proximal region of farA. farR3 is transcribed both as a monocistronic RNA and as a bicistronic farR4-farR3 mRNA, and the expression profile is tightly controlled by the IM-2/FarA system. Loss of farR3 delayed and decreased the production of indigoidine without any changes in the transcriptional profile of other far regulatory genes, indicating that FarR3 positively controls the biosynthesis of indigoidine and is positioned in the downstream region of the IM-2/FarA signaling system. Meanwhile, loss of farR4 induced the early production of IM-2 by increasing transcription of an IM-2 biosynthetic gene, farX, indicating that FarR4 negatively controls the biosynthesis of IM-2. Thus, our results suggested differential contributions of the SARP family regulators to the regulation of secondary metabolism in S. lavendulae FRI-5. This is the first report to show that an SARP family regulator is involved in the biosynthesis of a signaling molecule functioning at the most upstream region of the regulatory cascade for Streptomyces secondary metabolism.
Subject(s)
Biosynthetic Pathways/genetics , Gene Expression Regulation, Bacterial , Genes, Regulator , Piperidones/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Gene Expression Profiling , Pigments, Biological/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
To establish a high throughput and cost-efficiency procedure for distinguishing among cultivars in Lentinula edodes, the polymorphism in the genes reported previously in this fungus was examined using PCR or PCR-RFLP techniques with a high-efficiency genome scanning (HEGS) system. As a result, PCR-based markers derived from eight genes (tyr, cap, ppa, IGS-RFLP, pri B-RFLP, mfbC-RFLP, gla-RFLP, xy-RFLP) showed polymorphisms among cultivars in this fungus and consequently, enabled to distinguish seventy-nine cultivars used in this study.
Subject(s)
Genetic Markers , Genome, Fungal , Molecular Typing/methods , Mycological Typing Techniques , Polymerase Chain Reaction/methods , Shiitake Mushrooms/classification , Shiitake Mushrooms/genetics , Polymorphism, Genetic , Polymorphism, Restriction Fragment LengthABSTRACT
The gamma-butyrolactone signaling system is distributed widely among streptomycetes as an important regulatory mechanism of antibiotic production and/or morphological differentiation. IM-2 [(2R,3R,1'R)-2-(1'-hydroxybutyl)-3-hydroxymethyl-gamma-butanolide] is a gamma-butyrolactone that switches off the production of D: -cycloserine but switches on the production of several nucleoside antibiotics as well as blue pigment in Streptomyces lavendulae FRI-5. farX is a member of the afsA-family genes, which are proposed to encode enzymes involved in gamma-butyrolactone biosynthesis. Disruption of farX caused overproduction of D: -cycloserine, and abolished production of nucleoside antibiotic and blue pigment with the loss of IM-2 production. The finding that all phenotypic changes observed in the farX disruptant were restored by the addition of exogenous IM-2 suggested that FarX plays a biosynthetic role in IM-2 production. Transcriptional comparison between the wild-type strain and the farX disruptant revealed that, in addition to already known genes farR1 and farR2, several other genes (farR4, farD, and farE) are under the transcriptional regulation of IM-2. Furthermore, the fact that farX transcription is under the control of IM-2 suggested that S. lavendulae FRI-5 has a fine-tuning system to control gamma-butyrolactone production.
Subject(s)
4-Butyrolactone/biosynthesis , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial/physiology , Streptomyces/metabolism , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/genetics , 4-Butyrolactone/metabolism , Bacterial Proteins/genetics , Blotting, Northern , Cycloserine/metabolism , Gene Expression Regulation, Bacterial/genetics , Molecular Structure , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology , Streptomyces/geneticsABSTRACT
The gamma-butyrolactone-autoregulator signalling system is widely distributed across many Streptomyces species and it controls secondary metabolism and/or morphological differentiation. IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl-gamma-butanolide] is a gamma-butyrolactone autoregulator which, in Streptomyces lavendulae FRI-5, switches off the production of D-cycloserine, but switches on the production of several nucleoside antibiotics and blue pigment. In the IM-2 system, an IM-2 specific receptor (FarA) plays a critical role in the biosynthetic regulation of these metabolites, including IM-2 itself. Here, we identified five additional regulatory genes in the farA-flanking region and demonstrated that, in addition to farA, at least two more genes (farR1 and farR2) are involved in the IM-2/FarA system as the direct transcriptional target of FarA. The gel-shift assay revealed that FarA was bound to the upstream region of the four genes (including farR1 and farR2) in an IM-2-dependent manner. The FarA-binding sites were localized by DNase I footprinting to 27- to 33-bp palindromic structures, suggesting that FarA-binding sequences consist of two conserved hexamers separated by six nucleotides. Both farR1 and farR2 were transcribed in a growth-dependent manner, and marked expression was induced in the presence of IM-2, whereas transcripts of other two genes were not detected under the cultivation conditions used. The FarA-binding sites of farR1 and far2 overlap the promoter regions, suggesting that FarA represses the transcription of these two genes in the absence of IM-2 by inhibiting RNA polymerase access.
