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dc.contributor.authorMonson, RItaen
dc.contributor.authorSmith, Deborah Sen
dc.contributor.authorMatilla, Miguel Aen
dc.contributor.authorRoberts, Kevinen
dc.contributor.authorRichardson, Elizabethen
dc.contributor.authorDrew, Alisonen
dc.contributor.authorWilliamson, Neilen
dc.contributor.authorRamsay, Joshen
dc.contributor.authorWelch, Martinen
dc.contributor.authorSalmond, Georgeen
dc.date.accessioned2015-12-03T12:43:32Z
dc.date.available2015-12-03T12:43:32Z
dc.date.issued2015-12-03en
dc.identifier.citationFrontiers in Microbiology 2015, 6: 1442. doi:10.3389/fmicb.2015.01442en
dc.identifier.issn1664-302X
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/252825
dc.description.abstractRandom transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.
dc.description.sponsorshipThe authors would like to acknowledge several funding sources. D. Smith was supported by a PhD studentship from the BBSRC. Work in the MW lab is supported by the BBSRC (grants BB/G015171/1 and BB/M019411/1). K. Roberts was funded by an MRC studentship. R. Monson and the Salmond lab were supported by grants from the BBSRC (Grant No Provisional BB/K001833/1). M.A. Matilla was supported by the EU Marie-Curie Intra-European Fellowship for Career Development (FP7-PEOPLE-2011-IEF), grant number 298003. B. Richardson was supported by a Harry Smith vacation studentship from the SGM, UK. The authors would also like to thank Ray Chai for careful reading and comments on this manuscript. Alison Drew provided technical support. Work with plant pathogens was carried out under DEFRA licence No. 50864/197900/1.
dc.languageEnglishen
dc.language.isoenen
dc.publisherFrontiers
dc.rightsAttribution 2.0 UK: England & Wales*
dc.rights.urihttp://creativecommons.org/licenses/by/2.0/uk/*
dc.subjectplasposonen
dc.subjecttransposon mutagenesisen
dc.subjectenterobacteriaen
dc.subjectDickeyaen
dc.subjectSerratiaen
dc.subjectplant pathogenen
dc.subjectgas vesiclesen
dc.subjectoocydin Aen
dc.titleA plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteriaen
dc.typeArticle
dc.description.versionThis is the final version of the article. It was first available from Frontiers via http://dx.doi.org/10.3389/fmicb.2015.01442en
prism.number1442en
prism.publicationDate2015en
prism.publicationNameFrontiers in Microbiologyen
prism.volume6en
dc.rioxxterms.funderBBSRC
dc.rioxxterms.funderMRC
dc.rioxxterms.projectidBB/G015171/1
dc.rioxxterms.projectidBB/M019411/1
dc.rioxxterms.projectidBB/K001833/1
dcterms.dateAccepted2015-12-03en
rioxxterms.versionofrecord10.3389/fmicb.2015.01442en
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2015-12-03en
dc.contributor.orcidWelch, Martin [0000-0003-3646-1733]
dc.contributor.orcidSalmond, George [0000-0002-5197-2198]
dc.identifier.eissn1664-302X
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idBBSRC (BB/G015171/1)
pubs.funder-project-idBBSRC (BB/H013261/1)
pubs.funder-project-idBBSRC (BB/M019411/1)
pubs.funder-project-idBBSRC (BB/E015581/1)
pubs.funder-project-idBBSRC (BB/F009666/1)
pubs.funder-project-idBBSRC (BB/G000298/1)
pubs.funder-project-idBBSRC (BB/H002677/1)
pubs.funder-project-idEuropean Commission (298003)
pubs.funder-project-idBBSRC (BB/K001833/1)


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Attribution 2.0 UK: England & Wales
Except where otherwise noted, this item's licence is described as Attribution 2.0 UK: England & Wales