Construction of M. tuberculosis transposon libraries.

(A) The Mtb transposon mutagenesis library was generated from the siderophore-deficient mutant ML1600 (ΔmbtD::hyg^r). The library was cultured on low iron 7H9/ADS agar plates supplemented with (i) 500 ng/mL Fe-cMBT; (ii) 250 ng/mL Fe-MBT (high concentration); (iii) 50 ng/mL Fe-MBT (low concentration); (iv) 20 μM hemin; (v) 5 μM hemoglobin and (vi) 20 μM hemin plus 250 ng/mL Fe-MBT, respectively. The library DNA was isolated from the plates after three weeks. (B) Library building protocol. Genomic DNA pool was digested by HinP1I to generate GC-tails, allowing to the ligation of GC-tailed adapters which compose of short 3’ blocked and long oligonucleotides. The 3’ end of the shorter oligo was blocked (*) by C6-TFA-amino modification so that it could not be extended during amplification. The transposon-chromosome junctions were selectively amplified using primers (S2 Table) that recognize the end of the transposon and the 5’ end of the long adapter oligo. These primers contain all requisite sequences to permit direct multiplex sequencing of amplicons on an Illumina NextSeq 500 platform. (C) Transposon-chromosome junctions from high density mutant libraries were identified by deep sequencing. The six concentric circles in different colors represent the six libraries corresponding to (A). From the outer to the inner, they are (i) cMBT; (ii) high MBT; (iii) low MBT; (iv) hemin; (v) hemoglobin and (vi) hemin plus high MBT, respectively. The number of normalized sequence reads corresponding to each insertion site is represented as one color bars mapped onto the circular chromosome of M. tuberculosis H37Rv. Black contour represents the GC content of the chromosome (G+C content higher or lower than 50% are represented as contours outside or inside the ring, respectively). The genomic deletion regions such as ΔRD1, ΔpanCD and ΔmbtD are indicated in black. The genomic regions such as esx-3, irtA/B, mmpL5 and rv2047c are indicated in red. Nucleotide positions are indicated (million bases). Plots were generated using Circos 0.69–5 [43].