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...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-11-01
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| Series: | Animal Diseases |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s44149-021-00026-4 |
| _version_ | 1818750652388474880 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-18T04:23:05Z |
| format | Article |
| id | doaj.art-f094b55991ac4fa1b5e1c9c4b524d061 |
| institution | Directory Open Access Journal |
| issn | 2731-0442 |
| language | English |
| last_indexed | 2024-12-18T04:23:05Z |
| publishDate | 2021-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Animal Diseases |
| spelling | doaj.art-f094b55991ac4fa1b5e1c9c4b524d0612022-12-21T21:21:11ZengBMCAnimal Diseases2731-04422021-11-011111310.1186/s44149-021-00026-4Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)Janine T. Bossé0Yanwen Li1Leon G. Leanse2Liqing Zhou3Roy R. Chaudhuri4Sarah E. Peters5Jinhong Wang6Gareth A. Maglennon7Matthew T. G. Holden8Duncan J. Maskell9Alexander W. Tucker10Brendan W. Wren11Andrew N. Rycroft12Paul R. Langford13on behalf of the BRaDP1T consortiumSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s CampusSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s CampusSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s CampusSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s CampusDepartment of Veterinary Medicine, University of CambridgeDepartment of Veterinary Medicine, University of CambridgeDepartment of Veterinary Medicine, University of CambridgeDepartment of Pathology and Pathogen Biology, The Royal Veterinary CollegeThe Wellcome Trust Sanger InstituteDepartment of Veterinary Medicine, University of CambridgeDepartment of Veterinary Medicine, University of CambridgeFaculty of Infectious & Tropical Diseases, London School of Hygiene & Tropical MedicineDepartment of Pathology and Pathogen Biology, The Royal Veterinary CollegeSection of Paediatric Infectious Disease, Department of Infectious Disease, Imperial College London, St. Mary’s CampusAbstract 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.https://doi.org/10.1186/s44149-021-00026-4MarinerTransposonTraDISPasteurellaceaeActinobacillus pleuropneumoniaePasteurella multocida |
| spellingShingle | 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) Animal Diseases Mariner Transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida |
| title | 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_short | 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 |
| topic | Mariner Transposon TraDIS Pasteurellaceae Actinobacillus pleuropneumoniae Pasteurella multocida |
| url | https://doi.org/10.1186/s44149-021-00026-4 |
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