scholarly journals Identification and Utility of FdmR1 as a Streptomyces Antibiotic Regulatory Protein Activator for Fredericamycin Production in Streptomyces griseus ATCC 49344 and Heterologous Hosts

2008 ◽  
Vol 190 (16) ◽  
pp. 5587-5596 ◽  
Author(s):  
Yihua Chen ◽  
Evelyn Wendt-Pienkowski ◽  
Ben Shen
Keyword(s):  
Gene Cluster ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Streptomyces Griseus ◽  
Biosynthetic Gene Cluster ◽  
Positive Control ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Protein Activator ◽  
Biosynthetic Machinery

ABSTRACT The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

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 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 The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production

2007 ◽  
Vol 6 (7) ◽  
pp. 1210-1218 ◽  
Author(s):  
Daren W. Brown ◽  
Robert A. E. Butchko ◽  
Mark Busman ◽  
Robert H. Proctor
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Fusarium Verticillioides ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Regulatory Proteins ◽  
Wild Type ◽  
Polyketide Synthase Gene ◽  
Splice Forms ◽  
Self Protection

ABSTRACT Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Δfum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Δfum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

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 Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974

2009 ◽  
Vol 192 (2) ◽  
pp. 426-435 ◽  
Author(s):  
Silke I. Patzer ◽  
Volkmar Braun
Keyword(s):  
Gene Cluster ◽  
Linear Form ◽  
Streptomyces Griseus ◽  
Biosynthetic Gene Cluster ◽  
Peptide Structure ◽  
Biosynthetic Gene ◽  
Biosynthesis Gene Cluster ◽  
Streptomyces Sp ◽  
Peptide Synthetase ◽  
Biosynthesis Gene

ABSTRACT The main siderophores produced by streptomycetes are desferrioxamines. Here we show that Streptomyces sp. ATCC 700974 and several Streptomyces griseus strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the dhb genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the dhb genes. Heterologous expression of the dhb genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain Streptomyces lividans TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in Streptomyces.

scholarly journals Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1250-1259 ◽  
Author(s):  
Nattika Pulsawat ◽  
Shigeru Kitani ◽  
Eriko Fukushima ◽  
Takuya Nihira
Keyword(s):  
Gene Cluster ◽  
Response Regulator ◽  
Expression Profiles ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes ◽  
Streptomyces Virginiae

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

scholarly journals Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC

2005 ◽  
Vol 393 (1) ◽  
pp. 219-226 ◽  
Author(s):  
Eitaro Matsumura ◽  
Masashi Sakai ◽  
Katsuaki Hayashi ◽  
Shuichiro Murakami ◽  
Shinji Takenaka ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Gene Cluster ◽  
Specific Activity ◽  
Regulatory Protein ◽  
Constitutive Expression ◽  
Amino Acid Sequences ◽  
Upstream Region ◽  
Rhodococcus Species ◽  
Cat Gene ◽  
Rhodococcus Sp

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 °C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

scholarly journals REPRESSOR AND ANTIREPRESSOR IN THE REGULATION OF STAPHYLOCOCCAL PENICILLINASE SYNTHESIS

Genetics ◽  
1973 ◽  
Vol 75 (1) ◽  
pp. 1-17
Author(s):  
John Imsande
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Site ◽  
Regulatory Protein ◽  
Positive Control ◽  
Negative Control ◽  
Biochemical Data ◽  
Wild Type ◽  
Data Support ◽  
Operator Binding ◽  
Effector Binding

ABSTRACT 5-methyltryptophan (5MT) induces penicillinase synthesis in Staphylococcus aureus. The analog is incorporated into protein by both wild-type and tryptophan-starved cells. Since normal penicillinase repressor appears to contain tryptophan even though penicillinase itself does not, it is concluded that 5MT induces penicillinase synthesis by becoming incorporated into the penicillinase repressor and thereby inactivating the repressor. Thus biochemical data support the existence of a penicillinase repressor and indicate that penicillinase synthesis is regulated by negative control and not by positive control.—In the absence of exogenous tryptophan, staphylococcal penicillinase induction can be inhibited by 7-azatryptophan (7azaT). Because 7azaT is incorporated into protein by tryptophan-starved cells, it is concluded that 7azaT blocks penicillinase induction by inactivating a penicillinase regulatory protein into which the analog has been incorporated. Incorporation of 7azaT does not appear to inactivate the operator binding site or the effector binding site on the penicillinase repressor. Therefore, it appears that 7azaT blocks penicillinase induction by inactivating the penicillinase antirepressor, a protein required for inactivation of the penicillinase repressor and, hence, required for penicillinase induction.

Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus

1991 ◽  
Vol 229 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Dusica Vujaklija ◽  
Kenji Ueda ◽  
Soon-Kwang Hong ◽  
Teruhiko Beppu ◽  
Sueharu Horinouchi
Keyword(s):  
Gene Cluster ◽  
Streptomyces Griseus ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene
Sign in / Sign up

Export Citation Format

Share Document