Rationally designedmarinervectors to allow functional genomic analysis ofActinobacillus pleuropneumoniaeand other bacteria by transposon-directed insertion-site sequencing (TraDIS)

Abstract

Rationally designed mariner vectors to allow functional genomic analysis of 1 Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-2 site sequencing (TraDIS) 3 4 Abstract 29 30Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput 31 method for mapping insertion sites in large libraries of transposon mutants. The 32 Himar1 (mariner) transposon is ideal for generating near-saturating mutant 33 libraries, especially in AT-rich chromosomes, as the requirement for integration is 34 a TA dinucleotide. In this study, we generated two novel mariner vectors, 35 pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the 36 transposase gene), in order to facilitate TraDIS identification of conditionally 37 essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the 38 pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant 39 libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a 40 near random distribution of insertions around the respective chromosomes. A 41 preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, 42 that are required for growth under anaerobic conditions. 43 44 Importance 45 46 Comprehensive identification of conditionally essential genes requires 47 efficient tools for generating high-density transposon libraries that, ideally, can be 48 analysed using next-generation sequencing methods. The mariner transposon 49 has been used for mutagenesis of a wide variety of bacteria, however plasmids for 50 delivery of this transposon do not necessarily work well in all bacteria. In 51 particular, there are limited tools for functional genomic analysis of 52Pasteurellaceae species of major veterinary importance, such as swine and cattle 53 pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, 54 respectively. Here, we have developed plasmids that allow delivery of mariner and 55 the production of genome saturated mutant libraries for both of these pathogens, 56 but which should also be applicable to a wider range of bacteria. High-throughput 57 screening of the generated libraries will identify mutants required for growth under 58 different conditions, including in vivo, highlighting key virulence factors and 59 pathways that can be exploited for development of novel therapeutics and 60 vaccines. 61 62

Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum

“…To further remove any residual plasmid from the sequencing library, I-SceI recognition sites were incorporated into pMTL-CW20, which provided a mechanism for removal of plasmid reads at the sequencing library stage. After adapter ligation, an I-SceI restriction is used to cleave the site between the adapter and the library primer binding site on the transposon, making those fragments originating from plasmid DNA unsuitable templates for the subsequent PCR amplification step, as described in a similar strategy ( 27 ). Since there is no I-SceI recognition site in the C. autoethanogenum genome, transposon insertion sites in the genome will be identified as usual.…”

Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

“…To further remove any residual plasmid from the sequencing library I-SceI recognition sites were incorporated into pMTL-CW20 which provided a mechanism for removal of plasmid reads at the sequencing library stage. After adapter-ligation an I-SceI restriction is used to cleave the site between the adapter and the library primer binding site on the transposon, making those fragments originating from plasmid DNA unsuitable templates for the subsequent PCR amplification step as described in a similar strategy 26 . In the initial transposon library grown on rich medium, 0.2% of reads mapped to the transposon-delivery plasmid.…”