Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).
Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).
复制标题
DOI：
10.1186/s44149-021-00026-4
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
BRaDP1T consortium
中科院分区：
文献类型：
--
作者：
Bossé JT;Li Y;Leanse LG;Zhou L;Chaudhuri RR;Peters SE;Wang J;Maglennon GA;Holden MTG;Maskell DJ;Tucker AW;Wren BW;Rycroft AN;Langford PR;BRaDP1T consortium
条件必需基因的全面鉴定需要用于产生高密度转座子文库的有效工具，理想情况下，可以使用下一代测序方法如转座子定向插入位点测序（TraDIS）来分析所述转座子文库。Himar 1（mariner）转座子是产生近饱和突变体文库的理想选择，特别是在富含AT的染色体中，因为整合的要求是TA二核苷酸，并且该转座子已用于多种细菌的诱变。然而，用于水手递送的质粒不一定在所有细菌中都能很好地工作。特别是，存在有限的工具用于主要兽医重要性的巴斯德菌科物种的功能基因组分析，例如猪和牛病原体，分别为胸膜肺炎放线杆菌和多杀性巴斯德菌。在这里，我们开发了质粒pTsodCPC9和pTlacPC9（不同之处仅在于驱动转座酶基因表达的启动子），它们允许将mariner递送到这两种病原体中，但也应该适用于更广泛的细菌。使用pTlacPC9载体，我们首次在A.胸膜肺炎杆菌和多杀性巴氏杆菌，其显示如通过TraDIS检测的在各自染色体周围的插入的接近随机分布。初步筛选的5000个突变体，每个确定8和14个基因，分别是厌氧条件下生长所需的。未来对所产生的文库进行高通量筛选将有助于鉴定在不同条件下生长所需的突变体，包括体内，突出可用于开发新疗法和疫苗的关键毒力因子和途径。
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.