Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource

Abstract

Mutant 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 (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are 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 incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated 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 the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated. IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis 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 of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential 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.

Construction of an Ordered Transposon Library for Uropathogenic Proteus mirabilis HI4320

“…A close reading of the original ordered library technique revealed this group also identified “some frequency of picking multiple clones to the same well” due to bacterial clumping, and the authors suggested screening 5 to 10 colonies to obtain a pure clone ( 17 ). In a follow-up study, 11 to 14% of the wells of a Mycobacterium bovis library contained mixed clones ( 37 ). We estimate that the percentage is substantially higher in our library; based on the results in Fig.…”

“…This slow initial growth or small colony size did not necessarily correlate with the robustness of the mutant after restreaking. Borgers et al ( 37 ) found that human hands were more skilled at picking single colonies during library preparation and recommended against using a robot for picking colonies, as we used here. This issue is certainly species dependent, but it seems likely to hold true for P. mirabilis .…”

“…In future libraries, we would recommend (i) extensively troubleshooting mating parameters to select the best combination of single insertions and number of colonies ( 41 ); (ii) diluting the outputs (e.g., using more or larger plates [ 17 ]) to avoid picking mixed colonies, and (iii) fine-tuning postmating selection to eliminate breakthrough of the donor strain or choosing a mutagenesis method that avoids the need for a donor strain, such as transducing phage or electroporation of transposon/transposase complexes ( 37 , 42 ). Longer incubation of plates will also reveal if large numbers of smaller or slower-developing colonies and/or possible donor strain breakthrough events are likely to be forming within larger, earlier-developing colonies.…”

“…Alternatively, we could employ a modified transposon that would be more stable. For instance, ISceIM can be used to remove both the antibiotic resistance marker and sometimes one or both inverted repeats of the mariner transposon, making it unable to hop further ( 37 ). Future arrayed libraries could also incorporate dialable knockdown of message using technologies such as Mobile-CRISPRi, which shows promise but needs to be optimized for higher transfer efficiency in P. mirabilis ( 43 ).…”

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

“…Our biochemical data support the hypothesis that LamH is responsible for generating the capsular products of LM and LAM. To test this, we utilised a transposon mutant within lamH (BCGDAN_0378) in M. bovis BCG Danish, hereafter referred to as Δ lamH 30 . This species is 99% genetically identical to M. tuberculosis , having lost elements predominantly related to pathogenesis and is generally an excellent model system for M. tuberculosis envelope biogenesis and turnover.…”