Show simple item record

dc.contributor.authorBossé, Janine T
dc.contributor.authorLi, Yanwen
dc.contributor.authorLeanse, Leon G
dc.contributor.authorZhou, Liqing
dc.contributor.authorChaudhuri, Roy R
dc.contributor.authorPeters, Sarah E
dc.contributor.authorWang, Jinhong
dc.contributor.authorMaglennon, Gareth A
dc.contributor.authorHolden, Matthew TG
dc.contributor.authorMaskell, Duncan J
dc.contributor.authorTucker, Alexander W
dc.contributor.authorWren, Brendan W
dc.contributor.authorRycroft, Andrew N
dc.contributor.authorLangford, Paul R
dc.contributor.authorBRaDP1T consortium
dc.date.accessioned2021-12-15T10:08:16Z
dc.date.available2021-12-15T10:08:16Z
dc.date.issued2021
dc.date.submitted2021-07-05
dc.identifier.issn2731-0442
dc.identifier.others44149-021-00026-4
dc.identifier.other26
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/331438
dc.description.abstractComprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.
dc.languageen
dc.publisherSpringer Science and Business Media LLC
dc.subjectOriginal Article
dc.subjectMariner
dc.subjectTransposon
dc.subjectTraDIS
dc.subjectPasteurellaceae
dc.subjectActinobacillus pleuropneumoniae
dc.subjectPasteurella multocida
dc.titleRationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).
dc.typeArticle
dc.date.updated2021-12-15T10:08:16Z
prism.issueIdentifier1
prism.publicationNameAnim Dis
prism.volume1
dc.identifier.doi10.17863/CAM.78892
dcterms.dateAccepted2021-09-14
rioxxterms.versionofrecord10.1186/s44149-021-00026-4
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidBossé, Janine T [0000-0002-8491-4779]
dc.identifier.eissn2731-0442
pubs.funder-project-idBiotechnology and Biological Sciences Research Council (BB/G019274/1)
cam.issuedOnline2021-11-26


Files in this item

Thumbnail
Thumbnail

This item appears in the following Collection(s)

Show simple item record