Characterization of the YCjN ABC Transporter in Escherichia coli: Role in Maltose and Ethidium Bromide Transport.

Abstract

This study investigated the functional role of the ycjN gene in Escherichia coli, hypothesizing that its encoded protein, YcjN, functions as a carbohydrate transporter. Bioinformatics analysis confirmed YcjN as a solute-binding protein (SBP). The construction of a ΔycjN deletion strain revealed that ycjN is not essential for bacterial viability. Ethidium bromide (EB) accumulation and efflux assays demonstrated that both the overexpression strain (E. coli/pET28a-ycjN) and wild-type E. coli exhibited significantly higher intracellular EB fluorescence as compared to null-loaded and defective strains, indicating enhanced substrate uptake capacity. No significant differences in EB efflux rates were observed among the strains, confirming that ycjN encodes an inwardly directed uptake pump. Further experiments using ATPase inhibitors ortho-vanadate and CCCP revealed that the YcjNOPV transporter actively transports substrates via ATP hydrolysis, classifying it as an ATP-binding cassette (ABC) family transporter. Substrate specificity analysis using high-performance liquid chromatography (HPLC) and reducing sugar detection confirmed that YcjNOPV primarily transports maltose while also exhibiting glucoside transport activity. These findings establish the functional role of the ycjN gene in carbohydrate transport and provide a foundation for engineering applications involving ABC transporters.