Loss of SeqA confers low-level fluoroquinolone resistance through transcriptional reprogramming and RpoS dependence in E. coli.

SeqA is a key regulator of DNA replication initiation and chromosome cohesion in Escherichia coli. Loss of SeqA causes replication asynchrony, segregation defects, and growth delay, but its role in antibiotic susceptibility has remained unclear. Fluoroquinolones (FQs), which directly target bacterial DNA gyrase and topoisomerase IV to generate double-strand breaks (DSBs), provide a useful system to probe how chromosomal organization influences antibiotic response. In this study, we investigated whether SeqA loss alters sensitivity to FQs compared to antibiotics with non-DNA targets. MIC and MBC assays revealed that ΔseqA cells exhibit a specific low-level resistance to FQs, with ~1.5-fold higher inhibitory and bactericidal thresholds while retaining wildtype sensitivity to β-lactams and aminoglycosides. Using MuGam-GFP and RecA-GFP reporters, we showed that ΔseqA cells had fewer DSBs and mount an attenuated SOS response at wildtype MIC levels, enabling survival at otherwise lethal doses. Complementation restored wildtype sensitivity, confirming SeqA's direct involvement. Importantly, resistance was abolished in ΔseqA-rpoS double mutants and upon sub-MIC rifampicin treatment, demonstrating that RpoS-dependent transcriptional reprogramming underlies this phenotype. This suggested that ΔseqA strains acquire resistance through an RpoS-dependent regulatory effect that likely involves broad transcriptional reprogramming that underlies this phenotype. Together, these results showed that loss of SeqA alters chromosome organization in a way that lowers fluoroquinolone-induced DNA damage and enables RpoS-dependent low-level resistance.