An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35.
Identifieur interne : 000B51 ( Main/Exploration ); précédent : 000B50; suivant : 000B52An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35.
Auteurs : Tina Netzker [Allemagne] ; Volker Schroeckh [Allemagne] ; Matthew A. Gregory [Royaume-Uni] ; Michal Flak [Allemagne] ; Mario K C. Krespach [Allemagne] ; Peter F. Leadlay [Royaume-Uni] ; Axel A. Brakhage [Allemagne]Source :
- Applied and environmental microbiology [ 1098-5336 ] ; 2016.
Descripteurs français
- KwdFr :
- Antibactériens (métabolisme), Conjugaison génétique (MeSH), Délétion de gène (MeSH), Escherichia coli (génétique), Plasmides (métabolisme), Sirolimus (métabolisme), Streptomyces (génétique), Streptomyces (métabolisme), Techniques de knock-out de gènes (méthodes), Techniques de transfert de gènes (MeSH).
- MESH :
- génétique : Escherichia coli, Streptomyces.
- métabolisme : Antibactériens, Plasmides, Sirolimus, Streptomyces.
- méthodes : Techniques de knock-out de gènes.
- Conjugaison génétique, Délétion de gène, Techniques de transfert de gènes.
English descriptors
- KwdEn :
- MESH :
- chemical , metabolism : Anti-Bacterial Agents, Sirolimus.
- genetics : Escherichia coli, Streptomyces.
- metabolism : Plasmids, Streptomyces.
- methods : Gene Knockout Techniques.
- Conjugation, Genetic, Gene Deletion, Gene Transfer Techniques.
Abstract
UNLABELLED
Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.
IMPORTANCE
The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.
DOI: 10.1128/AEM.00371-16
PubMed: 27037115
PubMed Central: PMC4959151
Affiliations:
Links toward previous steps (curation, corpus...)
Le document en format XML
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Gregory</name><affiliation wicri:level="1"><nlm:affiliation>Isomerase Therapeutics, Ltd., Cambridge, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Isomerase Therapeutics, Ltd., Cambridge</wicri:regionArea><wicri:noRegion>Cambridge</wicri:noRegion></affiliation></author><author><name sortKey="Flak, Michal" sort="Flak, Michal" uniqKey="Flak M" first="Michal" last="Flak">Michal Flak</name><affiliation wicri:level="1"><nlm:affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation></author><author><name sortKey="Krespach, Mario K C" sort="Krespach, Mario K C" uniqKey="Krespach M" first="Mario K C" last="Krespach">Mario K C. Krespach</name><affiliation wicri:level="1"><nlm:affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation></author><author><name sortKey="Leadlay, Peter F" sort="Leadlay, Peter F" uniqKey="Leadlay P" first="Peter F" last="Leadlay">Peter F. Leadlay</name><affiliation wicri:level="4"><nlm:affiliation>Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.</nlm:affiliation><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Department of Biochemistry, University of Cambridge, Cambridge</wicri:regionArea><orgName type="university">Université de Cambridge</orgName><placeName><settlement type="city">Cambridge</settlement><region type="country">Angleterre</region><region type="région" nuts="1">Angleterre de l'Est</region></placeName></affiliation></author><author><name sortKey="Brakhage, Axel A" sort="Brakhage, Axel A" uniqKey="Brakhage A" first="Axel A" last="Brakhage">Axel A. Brakhage</name><affiliation wicri:level="1"><nlm:affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany axel.brakhage@leibniz-hki.de.</nlm:affiliation><country wicri:rule="url">Allemagne</country><wicri:regionArea>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena</wicri:regionArea><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion><wicri:noRegion>Jena</wicri:noRegion></affiliation></author></analytic><series><title level="j">Applied and environmental microbiology</title><idno type="eISSN">1098-5336</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Anti-Bacterial Agents (metabolism)</term><term>Conjugation, Genetic (MeSH)</term><term>Escherichia coli (genetics)</term><term>Gene Deletion (MeSH)</term><term>Gene Knockout Techniques (methods)</term><term>Gene Transfer Techniques (MeSH)</term><term>Plasmids (metabolism)</term><term>Sirolimus (metabolism)</term><term>Streptomyces (genetics)</term><term>Streptomyces (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>Antibactériens (métabolisme)</term><term>Conjugaison génétique (MeSH)</term><term>Délétion de gène (MeSH)</term><term>Escherichia coli (génétique)</term><term>Plasmides (métabolisme)</term><term>Sirolimus (métabolisme)</term><term>Streptomyces (génétique)</term><term>Streptomyces (métabolisme)</term><term>Techniques de knock-out de gènes (méthodes)</term><term>Techniques de transfert de gènes (MeSH)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Anti-Bacterial Agents</term><term>Sirolimus</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Escherichia coli</term><term>Streptomyces</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Escherichia coli</term><term>Streptomyces</term></keywords><keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Plasmids</term><term>Streptomyces</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Gene Knockout Techniques</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Antibactériens</term><term>Plasmides</term><term>Sirolimus</term><term>Streptomyces</term></keywords><keywords scheme="MESH" qualifier="méthodes" xml:lang="fr"><term>Techniques de knock-out de gènes</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Conjugation, Genetic</term><term>Gene Deletion</term><term>Gene Transfer Techniques</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Conjugaison génétique</term><term>Délétion de gène</term><term>Techniques de transfert de gènes</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>UNLABELLED</b></p><p>Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.</p></div><div type="abstract" xml:lang="en"><p><b>IMPORTANCE</b></p><p>The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.</p></div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">27037115</PMID><DateCompleted><Year>2017</Year><Month>09</Month><Day>18</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Electronic-Print"><Journal><ISSN IssnType="Electronic">1098-5336</ISSN><JournalIssue CitedMedium="Internet"><Volume>82</Volume><Issue>12</Issue><PubDate><Year>2016</Year><Month>06</Month><Day>15</Day></PubDate></JournalIssue><Title>Applied and environmental microbiology</Title><ISOAbbreviation>Appl Environ Microbiol</ISOAbbreviation></Journal><ArticleTitle>An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35.</ArticleTitle><Pagination><MedlinePgn>3481-3492</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1128/AEM.00371-16</ELocationID><Abstract><AbstractText Label="UNLABELLED">Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate.</AbstractText><AbstractText Label="IMPORTANCE">The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.</AbstractText><CopyrightInformation>Copyright © 2016, American Society for Microbiology. All Rights Reserved.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Netzker</LastName><ForeName>Tina</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Schroeckh</LastName><ForeName>Volker</ForeName><Initials>V</Initials><AffiliationInfo><Affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Gregory</LastName><ForeName>Matthew A</ForeName><Initials>MA</Initials><AffiliationInfo><Affiliation>Isomerase Therapeutics, Ltd., Cambridge, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Flak</LastName><ForeName>Michal</ForeName><Initials>M</Initials><AffiliationInfo><Affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Krespach</LastName><ForeName>Mario K C</ForeName><Initials>MKC</Initials><AffiliationInfo><Affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Leadlay</LastName><ForeName>Peter F</ForeName><Initials>PF</Initials><AffiliationInfo><Affiliation>Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Brakhage</LastName><ForeName>Axel A</ForeName><Initials>AA</Initials><AffiliationInfo><Affiliation>Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute (HKI), Jena, Germany axel.brakhage@leibniz-hki.de.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena, Germany.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2016</Year><Month>05</Month><Day>31</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Appl Environ Microbiol</MedlineTA><NlmUniqueID>7605801</NlmUniqueID><ISSNLinking>0099-2240</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D000900">Anti-Bacterial Agents</NameOfSubstance></Chemical><Chemical><RegistryNumber>W36ZG6FT64</RegistryNumber><NameOfSubstance UI="D020123">Sirolimus</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000900" MajorTopicYN="N">Anti-Bacterial Agents</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D003227" MajorTopicYN="N">Conjugation, Genetic</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D017353" MajorTopicYN="N">Gene Deletion</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D055786" MajorTopicYN="N">Gene Knockout Techniques</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D018014" MajorTopicYN="N">Gene Transfer Techniques</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010957" MajorTopicYN="N">Plasmids</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="N">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName 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