scholarly journals The Mithramycin Gene Cluster of Streptomyces argillaceus Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster

1999 ◽  
Vol 181 (2) ◽  
pp. 642-647 ◽  
Author(s):  
Felipe Lombó ◽  
Alfredo F. Braña ◽  
Carmen Méndez ◽  
José A. Salas
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Fold Increase ◽  
Open Reading Frames ◽  
Antibiotic Biosynthesis ◽  
Positive Regulators ◽  
Actinorhodin Production

ABSTRACT Sequencing of a 4.3-kb DNA region from the chromosome ofStreptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomycesantibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of themtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production byStreptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

scholarly journals Regulation of a Novel Gene Cluster Involved in Secondary Metabolite Production in Streptomyces coelicolor

2010 ◽  
Vol 192 (19) ◽  
pp. 4973-4982 ◽  
Author(s):  
Hindra ◽  
Patricia Pak ◽  
Marie A. Elliot
Keyword(s):  
Gene Cluster ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Genetic Locus ◽  
Regulatory Protein ◽  
Negative Control ◽  
Antibiotic Biosynthesis ◽  
Rnase Iii ◽  
Protein Encoding ◽  
Actinorhodin Production

ABSTRACT Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNA-encoding gene (α-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was α-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, α-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III.

scholarly journals Characterization of the Noncanonical Regulatory and Transporter Genes in Atratumycin Biosynthesis and Production in a Heterologous Host

Marine Drugs ◽  
2019 ◽  
Vol 17 (10) ◽  
pp. 560 ◽  
Author(s):  
Zhijie Yang ◽  
Xin Wei ◽  
Jianqiao He ◽  
Changli Sun ◽  
Jianhua Ju ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Negative Regulator ◽  
Over Expression ◽  
Lead Compounds ◽  
Mycobacteria Tuberculosis ◽  
Abc Transporter Genes

Atratumycin is a cyclodepsipeptide with activity against Mycobacteria tuberculosis isolated from deep-sea derived Streptomyces atratus SCSIO ZH16NS-80S. Analysis of the atratumycin biosynthetic gene cluster (atr) revealed that its biosynthesis is regulated by multiple factors, including two LuxR regulatory genes (atr1 and atr2), two ABC transporter genes (atr29 and atr30) and one Streptomyces antibiotic regulatory gene (atr32). In this work, three regulatory and two transporter genes were unambiguously determined to provide positive, negative and self-protective roles during biosynthesis of atratumycin through bioinformatic analyses, gene inactivations and trans-complementation studies. Notably, an unusual Streptomyces antibiotic regulatory protein Atr32 was characterized as a negative regulator; the function of Atr32 is distinct from previous studies. Five over-expression mutant strains were constructed by rational application of the regulatory and transporter genes; the resulting strains produced significantly improved titers of atratumycin that were ca. 1.7–2.3 fold greater than wild-type (WT) producer. Furthermore, the atratumycin gene cluster was successfully expressed in Streptomyces coelicolor M1154, thus paving the way for the transfer and recombination of large DNA fragments. Overall, this finding sets the stage for understanding the unique biosynthesis of pharmaceutically important atratumycin and lays the foundation for generating anti-tuberculosis lead compounds possessing novel structures.

scholarly journals Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 472-483 ◽  
Author(s):  
Ram P. Garg ◽  
Ronald J. Parry
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Positive Control ◽  
Biosynthetic Gene ◽  
Direct Repeats ◽  
Expression Plasmid ◽  
Intergenic Regions

Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the −35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA–vlmH and vlmI–vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.

scholarly journals Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

2015 ◽  
Vol 197 (15) ◽  
pp. 2536-2544 ◽  
Author(s):  
Letizia Lo Grasso ◽  
Sonia Maffioli ◽  
Margherita Sosio ◽  
Mervyn Bibb ◽  
Anna Maria Puglia ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Antibiotic Production ◽  
Regulatory Protein ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Mechanisms ◽  
Antibiotic Biosynthesis ◽  
Strain Atcc ◽  
Protein Coding ◽  
Content Type

ABSTRACTThe actinomyceteNonomuraeasp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by thedbvgene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation ofdbv6had no effect. In addition, overexpression ofdbv3led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons,dbv14-dbv8anddbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4,dbv29,dbv36, anddbv37) and of six operons (dbv2-dbv1,dbv14-dbv8,dbv17-dbv15,dbv21-dbv20,dbv24-dbv28, anddbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription ofdbv4and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation.IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomyceteNonomuraeasp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds.

scholarly journals Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

2004 ◽  
Vol 48 (9) ◽  
pp. 3468-3476 ◽  
Author(s):  
Miyuki Otsuka ◽  
Koji Ichinose ◽  
Isao Fujii ◽  
Yutaka Ebizuka
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Open Reading Frames ◽  
Modular Organization ◽  
Type I ◽  
Orf3 Protein ◽  
Polyketide Synthase Gene ◽  
Polyketide Chain ◽  
Reading Frames

ABSTRACT Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite.

scholarly journals Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from Rhodococcus opacus 1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence

1998 ◽  
Vol 180 (5) ◽  
pp. 1082-1094 ◽  
Author(s):  
Dirk Eulberg ◽  
Elena M. Kourbatova ◽  
Ludmila A. Golovleva ◽  
Michael Schlömann
Keyword(s):  
Gene Cluster ◽  
Regulatory Protein ◽  
Evolutionary Relationship ◽  
Sequence Divergence ◽  
Functional Adaptation ◽  
Open Reading Frames ◽  
Rhodococcus Opacus ◽  
Sequence Comparisons ◽  
Catabolic Enzymes ◽  
Chloromuconate Cycloisomerase

Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strainRhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of clcRbut separated from it by 222 bp, we detected the clcA andclcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R. opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.

scholarly journals Deletion of a regulatory gene within the cpk gene cluster reveals novel antibacterial activity in Streptomyces coelicolor A3(2)

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2343-2353 ◽  
Author(s):  
Marco Gottelt ◽  
Stefan Kol ◽  
Juan Pablo Gomez-Escribano ◽  
Mervyn Bibb ◽  
Eriko Takano
Keyword(s):  
Antibacterial Activity ◽  
Gene Cluster ◽  
Polyketide Synthase ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Feedback Mechanism ◽  
Biologically Active ◽  
Transcriptional Analysis ◽  
Type I ◽  
Negative Feedback Mechanism

Genome sequencing of Streptomyces coelicolor A3(2) revealed an uncharacterized type I polyketide synthase gene cluster (cpk). Here we describe the discovery of a novel antibacterial activity (abCPK) and a yellow-pigmented secondary metabolite (yCPK) after deleting a presumed pathway-specific regulatory gene (scbR2) that encodes a member of the γ-butyrolactone receptor family of proteins and which lies in the cpk gene cluster. Overproduction of yCPK and abCPK in a scbR2 deletion mutant, and the absence of the newly described compounds from cpk deletion mutants, suggest that they are products of the previously orphan cpk biosynthetic pathway in which abCPK is converted into the yellow pigment. Transcriptional analysis suggests that scbR2 may act in a negative feedback mechanism to eventually limit yCPK biosynthesis. The results described here represent a novel approach for the discovery of new, biologically active compounds.

Characterization of the LacI-type transcriptional repressor RbsR controlling ribose transport in Corynebacterium glutamicum ATCC 13032

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 150-164 ◽  
Author(s):  
Svenja S. Nentwich ◽  
Karina Brinkrolf ◽  
Lars Gaigalat ◽  
Andrea T. Hüser ◽  
Daniel A. Rey ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Regulatory Gene ◽  
Mrna Level ◽  
Regulatory Protein ◽  
Mobility Shift ◽  
Transcriptional Induction

The gene products of the rbsRACBD (rbs) operon of C. glutamicum (cg1410–cg1414) encode a ribose-specific ATP-binding cassette (ABC) transport system and its corresponding regulatory protein (RbsR). Deletion of the structural genes rbsACBD prohibited ribose uptake. Deletion of the regulatory gene rbsR resulted in an increased mRNA level of the whole operon. Analysis of the promoter region of the rbs operon by electrophoretic mobility shift assays identified a catabolite-responsive element (cre)-like sequence as the RbsR-binding site. Additional RbsR-binding sites were identified in front of the recently characterized uriR operon (uriR-rbsK1-uriT-uriH) and the ribokinase gene rbsK2. In vitro, the repressor RbsR bound to its targets in the absence of an effector. A probable negative effector of RbsR in vivo is ribose 5-phosphate or a derivative thereof, since in a ribokinase (rbsK1 rbsK2) double mutant, no derepression of the rbs operon in the presence of ribose was observed. Analysis of the ribose stimulon in the C. glutamicum wild-type revealed transcriptional induction of the uriR and rbs operons as well as of the rbsK2 gene. The inconsistency between the existence of functional RbsR-binding sites upstream of the ribokinase genes, their transcriptional induction during growth on ribose, and the missing induction in the rbsR mutant suggested the involvement of a second transcriptional regulator. Simultaneous deletion of the regulatory genes rbsR and uriR finally demonstrated a transcriptional co-control of the rbs and uriR operons and the rbsK2 gene by both regulators, RbsR and UriR, which were furthermore shown to recognize the same cognate DNA sequences in the operators of their target genes.

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