NextSeq 500 sequencing; TraDIS-Xpress finds mechanisms of action and synergies of a novel lipid IVA biosynthesis inhibitor
TraDIS-Xpress finds mechanisms of action and synergies of a novel lipid IVA biosynthesis inhibitor
The E. coli transposon mutant library used in this study was described by Yasir et al. (2020), and the S. Typhimurium library was described by Holden et al. (2022). The transposon-located promoter was induced with 1 mM IPTG in both E. coli and S. Typhimurium. For each condition, approximately 107 CFU/mL of the transposon mutant library was added to 10 mL LB broth supplemented with the corresponding concentration of antimicrobial. Each of the three analogues (003, 009 and 010) of OPT-2U1 were investigated at four concentrations around the MIC: ¼xMIC, ½xMIX, 1xMIC and 2xMIC, as well as an unstressed control. This corresponded to 8, 16, 32 and 64 µg/mL respectively. Conditions were then incubated for 24 hours at 37 ⁰C lightly shaken at 200 rpm. Following this, 1 mL of each culture was centrifuged to form a bacterial pellet. DNA was extracted from these pellets following the protocol outlined by Trampari et al. (2021). Genomic DNA was normalised to 11.1 ng/µL and tagmented using a MuSeek DNA fragment library preparation kit (ThermoFisher). Fragmented DNA was purified with AMPure XP beads (Beckman Coulter). DNA was amplified using primers customised for the tagmented ends of the DNA and biotinylated primers specific to transposon. Following another purification step, biotinylated fragmented DNA was incubated for 4 hours with streptavidin beads (Dynabeads® kilobaseBINDER™, Invitrogen) to capture only DNA fragments containing the transposon. A second PCR step using DNA bound to the beads used barcoded sequencing primers specific to the tagmented ends and to the transposon. Beads were removed from the reaction with a magnet and DNA was purified and size-selected using AMPure beads. Fragment length was quantified using a Tapestation (Aligent).