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.
Highlights
Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae, is the causative agent of porcine pleuropneumonia, a highly contagious, often fatal, respiratory disease that causes considerable economic losses to the swine industry worldwide (Sassu et al 2018)
Comparison of results for A. pleuropneumoniae indicated that the sodC promoter is constitutively expressed (Bossé et al 2009), isopropyl-ßD-galactopyranoside (IPTG) induction of C9 transposase expression from the lac promoter resulted in higher frequencies of transposition (10− 6–10− 8 compared to 10− 7–10− 10)
Subsequent Transposon Directed Insertion-site Sequencing (TraDIS) analysis of ISceI-digested A. pleuropneumoniae library DNA generated 16,565,883 sequence reads from the 5′ end of the mariner transposon
Summary
Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae, is the causative agent of porcine pleuropneumonia, a highly contagious, often fatal, respiratory disease that causes considerable economic losses to the swine industry worldwide (Sassu et al 2018). Tn10 has an insertion site preference for GCTNAGC (Bender and Kleckner 1992), and different insertional hotspots were reported in A. pleuropneumoniae STM studies (Fuller et al 2000a; Sheehan et al 2003; Bossé et al 2010), limiting the usefulness of this transposon for creating a fully saturating library. A more random transposon mutagenesis system in A. pleuropneumoniae is required to allow genome-wide analysis of fitness using high-throughput sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS) that precisely map, and quantitatively measure the relative abundance of each transposon insertion in a pool of mutants (Gawronski et al 2009; Langridge et al 2009; van Opijnen et al 2009)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
