Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transposition biases and strategies for novel mutant discovery

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99 % of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all ORFs do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096-derived transposons. Analysis of MycoMarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is the more adequate for generating a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases, with Tn5367 being the most skewed. These loci-dependent transposition biases led to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

Global assessment ofMycobacterium aviumsubspecieshominissuisgenetic requirement for growth and virulence

“…We aimed to find an MAH strain in which we could create high-density transposon mutant libraries. The transposon donor phagemid ϕMycoMarT7 is widely used (and recommended over Tn 5367 transposition [31]), for efficient mycobacterial transposon mutagenesis [16–20, 32]. ϕMycoMarT7 is derived from ϕAE87, which originates from mycobacteriophage TM4 [38, 39].…”

“…104 was the first MAH strain with a publicly available, fully assembled genome [29], and has thus naturally been widely studied, also by us (exemplified in [8, 10, 11, 29, 30]). MAH 104 is, however, resistant to transposon mutagenesis by ϕMycoMarT7, the phagemid preferred to generate high-density mycobacterial libraries [16–20, 31, 32]. Moreover, MAH 104 (along with many other Mav strains) transforms with low efficiency [28, 33–35], complicating the use of this strain in hypothesis-driven genetic manipulation.…”

Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne’s Disease

“…Himar1 -derived transposons have also been used to generate random mutant libraries in M. tuberculosis and MAP ( 131 – 134 ). The Himar1 transposon (MycoMarT7) was engineered into the same TM4 phasmid derivative described above.…”

“…Most of the MAP transposon mutant libraries were made with Tn 5367 ( 127 , 128 , 135 , 136 ) and fewer libraries have been constructed using the Himar1 -derived transposon MycoMarT7 ( 132 , 133 ). Our group has constructed a Himar1 library for MAP and used it to demonstrate random genomic insertions ( 134 ).…”

Transposon Mutagenesis in Mycobacterium avium Subspecies Paratuberculosis

“…More recently, our group compared the total genome insertions sites for the mycobacterial transposon Tn5367 with the mariner transposon MycoMarT7 carrying the Himar1 transposase [11]. The dinucleotide insertion recognition site (5 0 -TA-3 0 ) for MycoMarT7 was significantly more prevalent than the degenerate tetramer (5 0 -NNPy[A/T]A[A/T]NN-3 0 ) Tn5367 recognition sites such that only 37 genes do not contain a MycoMarT7 site, compared to 710 genes missing Tn5367 sites.…”