Article Figures & Data
Figures
- FIG 1
Creating markerless KOs from transposon mutants using an optimized Himar1 transposon. (a) Visual representation of the original Himar1 transposon (2,064 bp), which consists of a kanamycin resistance marker (KanR) (795 bp) and an origin of replication for E. coli (OriE) (732 bp) flanked by two inverted repeats (IR) (27 bp). (b) Optimized Himar1 transposon (3,980 bp), which consists of a KanR (795 bp), an OriE (614 bp), and a SacB negative selection marker (1,419 bp) flanked by two FRT sites (34 bp), two I-SceI sites (18 bp), and two IRs (27 bp). (c) Illustration of the construction of a transposon mutant (marked) using the optimized transposon and subsequent unmarking strategies by bringing FlpE or ISceIM into the cells and selecting the unmarked mutants. FlpE/FRT-mediated recombination inherently leaves an FRT scar (IR-I-SceI-FRT-I-SceI-IR; 166 bp) in the unmarked transposon mutant, whereas ISceIM-based transposon removal has the potential of creating KO mutants free of heterologous DNA. (d and e) Proof of concept of the unmarking of three M. bovis BCG Danish transposon mutants (I, II, and III). Transposon mutants were unmarked via FlpE/FRT-mediated recombination (d) or ISceIM-based transposon removal (e). Therefore, replicable vectors expressing FlpE or ISceIM were introduced in the cells via electroporation, after which unmarked mutants (kanamycin sensitive and sucrose resistant) were analyzed via colony PCR. Parental transposon mutants (I, II, and II) and the original wild-type (WT) strain were taken along as controls. Lengths of expected amplicons are indicated in blue in panels c, d and e. x, length of WT amplicon; tn, transposon. Used primers are listed in Table S1 at https://doi.org/10.6084/m9.figshare.c.4507472.
- FIG 2
Unmarking M. smegmatis and M. bovis BCG Danish transposon mutants using different genetic carriers bringing FlpE or ISceIM to expression. Two or three transposon mutants for M. smegmatis (top) or M. bovis BCG Danish (bottom) were unmarked by introducing FlpE or ISceIM by different genetic carriers, expressing FlpE or ISceIM from a constitutive (const.) or inducible promoter. The efficiency of transformation (amount of hygromycin-resistant clones) was given a score ranging from - to +++ based on the observed CFU after incubation at 37°C (unless otherwise indicated, e.g., for the ts vectors). The efficiency of unmarking is indicated by the percentage of unmarked clones (kanamycin sensitive and sucrose resistant) among the sucrose-resistant clones (and hygromycin resistant for replicable vectors) obtained after unmarking. The efficiency of curing is indicated by the percentage of cured clones (hygromycin sensitive). All experiments were replicated once or more (e.g., replicable vectors), except the data for the replicable GalK vectors with the Tet-inducible promoters, as no improvement was seen in the transformation efficiency compared to that of the replicable GalK vectors with the constitutive promoters in the first experiment. NA, not applicable (vector or phage cannot replicate in mycobacteria); /, not performed (would not give any added value); std, standard; GalK, negative selection marker galactokinase; ts, temperature sensitive; acet. ind., acetamide inducible; tet-ind., tetracycline inducible; *, problem with the use of the ts vectors in BCG Danish: BCG Danish did not grow at 32°C but grew at 39°C. In gray, we indicate the most preferred tools.
- FIG 3
Outcome of unmarking transposon (tn) mutants via the I-SceI system. (a) Graphic illustration of the outcome of unmarking via the I-SceI system. Upon introduction of ISceIM, the restriction enzyme will cut the I-SceI sites, creating a DSB that will be repaired by NHEJ, resulting in one of three possible outcomes. (b and c) The outcome of the I-SceI system was analyzed for three M. smegmatis transposon mutants (b) and two BCG Danish transposon mutants (c) by performing PCR on kanamycin-sensitive and sucrose-resistant clones and Sanger sequencing the PCR amplicons (for M. smegmatis, only a subset of the clones was Sanger sequenced). Outcome is given in percentages. Used primers are listed in Table S1 at https://doi.org/10.6084/m9.figshare.c.4507472.
- FIG 4
Results of CP-CSeq on a transposon insertion library of M. bovis BCG Danish (2 sets of 96- by 96-well plates, set I and set II [this study]) compared to M. bovis BCG Pasteur (9). (a) Number of traceable transposon insertions for each library (set). (b) Distribution between disrupted genes that are traceable (purple), disrupted genes for which the locations of their disruption mutants are unknown (“untraceable” [gray]), and untargeted genes or genes in duplicated regions (brown). (c) Percentage of targeted nonessential genes for each library (set). (d) Nonessential genome saturation in function of the amount of traceable mutants. In green are the observed values for the library in M. bovis BCG Pasteur (P). In blue are the observed values for the library in M. bovis BCG Danish (set I, set II, and set I plus set II). To model what would be gained by further enlargement of CP-CSeq libraries, we fitted a coupon collector’s equation (in black) to the available experimental data from our libraries, which should allow more accurate predictions in the future. The coupon collector’s problem estimates the amount of traceable mutants needed to target a certain percentage of the nonessential genome. The equation for the curve is given in the graph. This curve goes not through 100%, probably due to inherent characteristics of transposon mutagenesis and clone picking of transposon mutants grown on agar.
- FIG 5
Positional validation of a subset of transposon insertion mutants by PCR. Ninety CP-CSeq assignments for transposon insertion mutants coming from 4 different 96-well plates were verified by PCR with a primer hybridizing to the transposon IR and a gene-specific primer. For plates I-1, II-33, and II-81, containing <96 mutants, we selected 18 transposon insertion mutants with a coverage of >300. For plate I-65, containing >96 mutants, we selected 18 transposon insertion mutants with a coverage of >1,200 (I-65*) and 18 transposon insertion mutants with a coverage of >300 and/or in wells with >1 mutant (I-65**). All CP-CSeq assignments were confirmed, except for 1 from plate II-81 (***). This PCR was successfully redone on a fraction of the glycerol stock (small gel on the right), so that all CP-CSeq assignments were confirmed. The list of mutants and their corresponding primers can be found in Table S3 at https://doi.org/10.6084/m9.figshare.c.4507472.
- FIG 6
Applications of transposon mutagenesis using the optimized transposon. Shown is an overview of how an archived transposon library can be generated and how transposon mutants of interest can be isolated and unmarked. The scheme includes the different applications (in blue) of the generated products. MTBC, Mycobacterium tuberculosis complex.
