Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

Abstract Comprehensive 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...

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Autores principales:	Janine T. Bossé, Yanwen Li, Leon G. Leanse, Liqing Zhou, Roy R. Chaudhuri, Sarah E. Peters, Jinhong Wang, Gareth A. Maglennon, Matthew T. G. Holden, Duncan J. Maskell, Alexander W. Tucker, Brendan W. Wren, Andrew N. Rycroft, Paul R. Langford, on behalf of the BRaDP1T consortium
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Mariner Transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida Veterinary medicine SF600-1100 Public aspects of medicine RA1-1270
Acceso en línea:	https://doaj.org/article/f094b55991ac4fa1b5e1c9c4b524d061
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spelling	oai:doaj.org-article:f094b55991ac4fa1b5e1c9c4b524d0612021-11-28T12:03:57ZRationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)10.1186/s44149-021-00026-42731-0442https://doaj.org/article/f094b55991ac4fa1b5e1c9c4b524d0612021-11-01T00:00:00Zhttps://doi.org/10.1186/s44149-021-00026-4https://doaj.org/toc/2731-0442Abstract Comprehensive 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.Janine T. BosséYanwen LiLeon G. LeanseLiqing ZhouRoy R. ChaudhuriSarah E. PetersJinhong WangGareth A. MaglennonMatthew T. G. HoldenDuncan J. MaskellAlexander W. TuckerBrendan W. WrenAndrew N. RycroftPaul R. Langfordon behalf of the BRaDP1T consortiumBMCarticleMarinerTransposonTraDISPasteurellaceaeActinobacillus pleuropneumoniaePasteurella multocidaVeterinary medicineSF600-1100Public aspects of medicineRA1-1270ENAnimal Diseases, Vol 1, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Mariner Transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida Veterinary medicine SF600-1100 Public aspects of medicine RA1-1270
spellingShingle	Mariner Transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida Veterinary medicine SF600-1100 Public aspects of medicine RA1-1270 Janine T. Bossé Yanwen Li Leon G. Leanse Liqing Zhou Roy R. Chaudhuri Sarah E. Peters Jinhong Wang Gareth A. Maglennon Matthew T. G. Holden Duncan J. Maskell Alexander W. Tucker Brendan W. Wren Andrew N. Rycroft Paul R. Langford on behalf of the BRaDP1T consortium Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
description	Abstract Comprehensive 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.
format	article
author	Janine T. Bossé Yanwen Li Leon G. Leanse Liqing Zhou Roy R. Chaudhuri Sarah E. Peters Jinhong Wang Gareth A. Maglennon Matthew T. G. Holden Duncan J. Maskell Alexander W. Tucker Brendan W. Wren Andrew N. Rycroft Paul R. Langford on behalf of the BRaDP1T consortium
author_facet	Janine T. Bossé Yanwen Li Leon G. Leanse Liqing Zhou Roy R. Chaudhuri Sarah E. Peters Jinhong Wang Gareth A. Maglennon Matthew T. G. Holden Duncan J. Maskell Alexander W. Tucker Brendan W. Wren Andrew N. Rycroft Paul R. Langford on behalf of the BRaDP1T consortium
author_sort	Janine T. Bossé
title	Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
title_short	Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
title_full	Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
title_fullStr	Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
title_full_unstemmed	Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)
title_sort	rationally designed mariner vectors for functional genomic analysis of actinobacillus pleuropneumoniae and other pasteurellaceae species by transposon-directed insertion-site sequencing (tradis)
publisher	BMC
publishDate	2021
url	https://doaj.org/article/f094b55991ac4fa1b5e1c9c4b524d061
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Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

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