Sequencing of ponA variant pools in Neisseriae gonorrhoeae under penicillin selection

This project examines the evolutionary dynamics and competitive fitness of engineered Neisseria gonorrhoeae strains carrying amino acid substitutions at residue 421 of penicillin-binding protein 1 (PBP1), encoded by ponA. Sixteen isogenic mutants were constructed in the genetic background of FA6140, a penicillin-resistant clinical isolate. Each mutant encodes a distinct amino acid at position 421, enabling systematic investigation of how variation at this key residue influences Beta-lactam tolerance and growth.The 16 FA6140-derived mutants were pooled in equal proportions and serially passaged in vitro under four penicillin concentrations (0, 0.25, 0.5, and 1.0 ug/mL) for 36 hours. Mixed genomic DNA was collected at every 6 hour time-point to quantify how antibiotic exposure shapes the relative abundance of ponA variants during growth.Two sequencing strategies were used to resolve population dynamics. Targeted amplicon sequencing of ponA was performed on time-point-specific DNA to precisely measure codon frequencies at residue 421 across the pooled populations. In parallel, whole-genome sequencing was conducted on genomic DNA collected at hour 0 and hour 36 from each antibiotic condition. These metagenomic WGS datasets represent pooled genomes of the strains present at each endpoint and provide context for genomic background stability.Together, these datasets characterize how specific PBP1 substitutions affect growth during antibiotic challenge and illuminate the selective forces acting on ponA in a clinically relevant, penicillin-resistant background. The results contribute to our understanding of chromosomally mediated Beta-lactam resistance and evolutionary potential in N. gonorrhoeae, supporting the associated publication on ponA variant dynamics under penicillin selection.