Transposon Sequencing of Bacterial Strains

TnSeq was conducted as described before [42 (link),43 (link)]; Briefly, cultures of strains MA1 and MA73 were mated with donor strain E.coli MFD λ pir, which contains the pSC189 suicide plasmid encoding the mariner transposon. Approximately 200,000 colonies/replicate were recovered from plates containing Kan50 to select for transposon insertions and 1mM IPTG to allow the expression of ShyA^L109K. Libraries were then resuspended in 30 mL of LB broth, and 1/5 of the culture was used for genomic DNA (gDNA) extraction and the rest was frozen in 30% glycerol at -80°C. Samples were prepared for sequencing as follows. The extracted gDNA was sheared by sonication (9 seconds, 30% amplitude), followed by blunting (Blunting Enzyme Mix, NEB), A-tailing, ligation of specific adaptors and PCR amplification of the transposon-DNA junctions using transposon- and adaptor- specific primers. Libraries were sequenced using Illumina MiSeq as described previously [44 (link)]. To determine gene essentiality, data analysis was performed using the Matlab-based pipeline ARTIST [43 (link)]. Genetic regions predicted to be conditionally essential/enriched were inspected using the genome browser Artemis [45 (link)] and insertion plots were generated using the tidyverse package in R.