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Construction and Use of Transposon MycoTetOP(2) for Isolation of Conditional Mycobacteria Mutants

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dc.contributor.authorRiggs-Shute, Sarah D.en
dc.contributor.authorFalkinham, Joseph O. IIIen
dc.contributor.authorYang, Zhaominen
dc.contributor.departmentBiological Sciencesen
dc.date.accessioned2020-05-22T13:55:41Zen
dc.date.available2020-05-22T13:55:41Zen
dc.date.issued2020-01-21en
dc.description.abstractMycobacteria are unique in many aspects of their biology. The development of genetic tools to identify genes critical for their growth by forward genetic analysis holds great promises to advance our understanding of their cellular, physiological and biochemical processes. Here we report the development of a novel transposon, MycoTetOP(2), to aid the identification of such genes by direct transposon mutagenesis. This mariner-based transposon contains nested anhydrotetracycline (ATc)-inducible promoters to drive transcription outward from both of its ends. In addition, it includes the Escherichia coli R6K gamma origin to facilitate the identification of insertion sites. MycoTetOP(2) was placed in a shuttle plasmid with a temperature-sensitive DNA replication origin in mycobacteria. This allows propagation of mycobacteria harboring the plasmid at a permissive temperature. The resulting population of cells can then be subjected to a temperature shift to select for transposon mutants. This transposon and its delivery system, once constructed, were tested in the fast-growing model Mycobacterium smegmatis and 13 mutants with ATc-dependent growth were isolated. The identification of the insertion sites in these mutants led to nine unique genetic loci with genes critical for essential processes in both M. smegmatis and Mycobacterium tuberculosis. These results demonstrate that MycoTetOP(2) and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.en
dc.description.notesSR-S received support from the Department of Biological Sciences at Virginia Tech. ZY received funding from National Institutes of Health grant GM071601 and National Science Foundation grant MCB1919455. We acknowledge Virginia Tech Open Access Subvention Fund for partial defrayment of the cost for the publication of this article.en
dc.description.sponsorshipDepartment of Biological Sciences at Virginia Tech; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM071601]; National Science FoundationNational Science Foundation (NSF) [MCB1919455]; Virginia Tech Open Access Subvention Funden
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.3389/fmicb.2019.03091en
dc.identifier.issn1664-302Xen
dc.identifier.other3091en
dc.identifier.pmid32038540en
dc.identifier.urihttp://hdl.handle.net/10919/98530en
dc.identifier.volume10en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjecttransposonen
dc.subjectmycobacteriumen
dc.subjectessential genesen
dc.subjectconditional mutantsen
dc.subjectTetR-tetOen
dc.titleConstruction and Use of Transposon MycoTetOP(2) for Isolation of Conditional Mycobacteria Mutantsen
dc.title.serialFrontiers in Microbiologyen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.dcmitypeStillImageen

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