Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone orderedM. bovisBCG mutant resource

AbstractMutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archivedMycobacteriumtransposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporatingFRTsites for FlpE/FRT-mediated recombination andI-SceIsites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. TheFRTapproach is highly efficient but leaves anFRTscar in the genome, whereas theI-SceImediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of theM. tbcomplex (18,432 clones, mutating 83% of the non-essentialM. tbhomologues), from which clean knockouts can be generated.ImportanceWhile speeding up research for many fields of biology (e.g. yeast, plant, andC. elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain ofM. bovisBCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed upMycobacteriumresearch (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of theM. tbcomplex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.