Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ

To streamline the elucidation of antibacterial compounds’ mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing.

These master plates were grown stationary at 37 o C for 48 hours.
For primary screening, cultures in the master plates were robotically inoculated into 96-well plates containing LB with 100 μg/mL trimethoprim and with or without 0.2% rhamnose. After 16 hours of incubation without shaking at 37 o C, the conditional growth phenotype was assessed as 50% or less growth (by OD600nm) without rhamnose.
Putative conditional growth phenotypes were validated by secondary screening for growth with or without rhamnose as before. Mutants that passed both screens (approximately one third of the initial clones) were stocked in LB with 20% glycerol and kept at -80 o C. The transposon insertion sites were determined by Tn-seq circle (1,2).
The redundant knockdown mutant library (830 clones) and 134 previously obtained knockdown mutants (3) were grown in 96-well format in LB with 0.2% rhamnose and 100 μg/mL trimethoprim at 37 o C overnight. The following day, mutants were pooled in equal amounts by OD600nm, forming the combined knockdown mutant library. Pool aliquots were stored in LB with 20% glycerol at -80 o C until needed.

Competitive Fitness Assay and Sequencing Data Analysis
For culturing, all the knockdown mutants were inoculated into 5 mL LB to a final OD600nm of 0.0025 with individual mutants at an approximate OD600nm of 2.5 X•10 -6 and with or without C109 or novobiocin, added at a concentration that inhibited 25% of K56-2 growth (IC25, 2.5 and 2 μg/mL, respectively). Rhamnose was added at the sensitizing concentration of 0.05%, which produced 30% to 60% of wild-type growth. The cultures were grown for 20 hours (approximately 20 generations) at 37 o C with 230 rpm shaking.
Mutants that were recovered after growth without antibiotics are shown in Table S1.
Wild-type B. cenocepacia K56-2 controls to check for 25% growth inhibition, and single mutant cultures to check for 30-60% wild-type growth were set up alongside the mutant pools and assessed by OD600nm after 20 hours. Cultures were harvested, the genomic DNA was isolated, and the Tn-seq circle method were performed as previously described (1,2). PCR primers 681, 690, 715, 717, 718, 719, 729 contain the Nextera indices and were used in appropriate pairs (Table S5). Indexed samples were pooled and sequenced at Génome Québec with an Illumina HiSeq 2500. Raw reads were deposited in the NCBI Sequence Read Archive (SRA) repository and will be publicly available after publication under accession SRP148709. All custom scripts used for data processing can be found at https://github.com/mdomarat/CardonaLab. The DNA reads were trimmed (filter_reads.py) and mapped (map_reads.py) to the contigs of the K56-2 draft genome (1). Insertion sites were then called and annotated with affected genes (identify.py). To compare the C109-or novobiocin-treated samples to the no antibiotic controls, insertion sites were merged based on position (expression.py). All insertion sites with fewer than 1000 reads in the no antibiotic controls were removed from the analysis. Reads were then normalized by total read count. Significance was assessed by calculating P-values as per Pierce et al. (4). Log2(Depletion) values for each mutant was then calculated as the log2 ratio of the average normalized reads in the no antibiotic control to those of the antibiotic-treated sample. Log2(Depletion) values of mutants that passed the significance threshold of P-value < 0.05 were fit to a normal distribution (5) and candidate targets were taken as greater than two standard deviations from the mean.          All values are reported as the mean of three biological replicates Table S4. Bacterial strains and plasmids used in this work.

Strain or plasmid Features Source
Burkholderia. cenocepacia