Identification of the active mechanism of aminoglycoside entry inV. choleraethrough characterization of sRNActrR,regulating carbohydrate utilization and transport

Abstract

AbstractThe possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism inVibriospecies. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment ofV. choleraewith sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNAncRNA586during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin. This sRNA is located near sugar transport genes and previous work on a homologous region inVibrio tasmaniensissuggested that this sRNA stabilizes gene transcripts for carbohydrate transport and utilization, as well as phage receptors. The role forncRNA586, hereafter namedctrR, in the transport of both carbohydrates and aminoglycosides, was further investigated. Flow cytometry on cells treated with a fluorescent aminoglycoside confirmed the role ofctrRand of carbohydrate transporters in differential aminoglycoside entry. Despite sequence diversity,ctrRshowed functional conservation across the Vibrionales. This system in directly modulated by carbon sources, suggesting regulation by carbon catabolite repression, a widely conserved mechanism in Gram-negative bacteria, priming future research on aminoglycoside uptake by sugar transporters in other bacterial species.