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      A Novel TetR Family Transcriptional Regulator, CalR3, Negatively Controls Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882

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          Abstract

          Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (Δ calR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in calcimycin biosynthesis and suggest that calcimycin production can be improved by manipulating its biosynthetic machinery.

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          Most cited references 27

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          Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument.

          We have adapted the techniques of DNA footprint analysis to an Applied Biosystems 3730 DNA Analyzer. The use of fluorescently labeled primers eliminates the need for radioactively labeled nucleotides, as well as slab gel electrophoresis, and takes advantage of commonly available automated fluorescent capillary electrophoresis instruments. With fluorescently labeled primers and dideoxynucleotide DNA sequencing, we have shown that the terminal base of each digested fragment may be accurately identified with a capillary-based instrument. Polymerase chain reaction (PCR) was performed with a 6FAM-labeled primer to amplify a typical target promoter region. This PCR product was then incubated with a transcriptional activator protein, or bovine serum albumin as a control, and then partially digested with DNase I. A clone of the promoter was sequenced with the Thermo Sequenase Dye Primer Manual Cycle Sequencing kit (USB) and the FAM-labeled primer. Through the use of Genemapper software, the Thermo sequenase and DNasei digestion products were accurately aligned, providing a ready means to assign correct nucleotides to each peak from the DNA footprint. This method was used to characterize the binding of two different transcriptional activator proteins to their respective promoter regions.
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            Initiation of actinorhodin export in Streptomyces coelicolor.

            Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.
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              Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor.

              We used the Tn10-encoded Tet repressor, which has a highly specific binding capacity for tetracycline, to probe contacts between the drug and protein by chemical interference studies of the antibiotic. For that purpose, the equilibrium association constants of modified tetracyclines with the Tet repressor and Mg2+ cations were determined quantitatively. The results confirm the previous notion that Mg2+ probably binds with the oxygens at positions 11 and 12 and is absolutely required for protein-drug recognition. Modifications were introduced at positions seven, six, five, and four of the drug, and anhydrotetracycline was also studied. Substitutions or eliminations of functions at these positions influenced binding to the Tet repressor up to 35-fold. The introduction of an azido function at position seven in 7-azidotetracycline and epimerization of the substituents at position four in 4-epitetracycline lead to a 2- or 25-fold reduction, respectively, of Tet repressor affinity in those compounds. Anhydrotetracycline bound about 35-fold more strongly than tetracycline did, indicating that the oxygen at position 11 may be involved in Tet repressor recognition. This increased binding is in contrast to the lower antibiotic activity of anhydrotetracycline and indicates that the Tet repressor and ribosomes recognize the drug differently.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                29 November 2017
                2017
                : 8
                Affiliations
                1School of Life Sciences, North China University of Science and Technology , Tangshan, China
                2Hebei Province Key Laboratory of Occupational Health and Safety for Coal Industry, School of Public Health, North China University of Science and Technology , Tangshan, China
                3School of Pharmacy, North China University of Science and Technology , Tangshan, China
                4State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai, China
                Author notes

                Edited by: Weiwen Zhang, Tianjin University, China

                Reviewed by: Wei Ding, Lanzhou University, China; Yinhua Lu, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences (CAS), China

                *Correspondence: Tiesheng Han, ts_han@ 123456163.com Zhijun Wang, wangzhijun@ 123456sjtu.edu.cn

                This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2017.02371
                5712553
                Copyright © 2017 Gou, Han, Wang, Ge, Liu, Hu and Wang.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 7, Tables: 1, Equations: 0, References: 32, Pages: 10, Words: 0
                Funding
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Award ID: 31700083
                Funded by: Natural Science Foundation of Hebei Province 10.13039/501100003787
                Award ID: C2015209051
                Award ID: H2015209094
                Categories
                Microbiology
                Original Research

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