17
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

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

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          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.

          Related collections

          Most cited references27

          • Record: found
          • Abstract: found
          • Book: not found

          Molecular Cloning : A Laboratory Manual

          <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            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.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways.

              Ca(2+) ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3(-), a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca(2+) increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.
                Bookmark

                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
                : 2371
                Affiliations
                [1] 1School of Life Sciences, North China University of Science and Technology , Tangshan, China
                [2] 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
                [3] 3School of Pharmacy, North China University of Science and Technology , Tangshan, China
                [4] 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
                29238338
                a9bbc18c-f261-4250-a8dc-1651ecb670d8
                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.

                History
                : 21 September 2017
                : 16 November 2017
                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

                Microbiology & Virology
                tetr family regulator,calr3 ligands,biosynthesis,calcimycin,streptomyces chartreusis

                Comments

                Comment on this article