Transposon mutagenesis uncovers the genetic landscape of streptomycin susceptibility and implicates SbmA in aminoglycoside uptake in Escherichia coli

Aminoglycosides are critical antibiotics with partially elucidated mechanisms of uptake and action in Gram-negative bacteria. Importantly, although energy-dependent uptake across the inner membrane has been well-established, the specific molecular mechanisms involved have not been definitively identified. To deepen understanding of genetic factors influencing susceptibility and resistance to streptomycin, we applied transposon insertion sequencing in Escherichia coli K-12. This approach identified both known and novel genes whose disruption increased susceptibility, including those involved in respiration, protein export, cell division, and uncharacterised functions. Notably, voltage-sensitive membrane dye-based assays revealed that many susceptible mutants did not display inner membrane hyperpolarisation as often assumed. Conversely, disruption of certain genes, such as the inner membrane antimicrobial peptide transporter sbmA , conferred low-level resistance, with sbmA overexpression increasing streptomycin sensitivity, suggesting its role in aminoglycoside uptake. These findings refine the model of aminoglycoside interaction with various pathways and highlight potential targets for adjuvant therapies to combat antimicrobial resistance.