Deciphering a missing piece of the branched-chain amino acids uptake puzzle: YhjE is an L-isoleucine and L-valine transporter in Escherichia coli K-12

Branched-chain amino acid (BCAA) uptake in bacteria underpins fitness, pathogenesis, and industrial strain performance, yet the transport landscape, even in <i>Escherichia coli</i>, remains incomplete. Here, we identify and characterize YhjE as a BCAA permease specific for L-isoleucine and L-valine. Selection for L-valine-resistant clones in a <i>ΔlivKHMGF ΔbrnQ</i> background repeatedly recovered mutations in <i>yhjE</i>, indicating that YhjE mediates L-valine entry. Deletion of <i>yhjE</i> in the <i>ΔlivKHMGF ΔbrnQ</i> background increased the L-isoleucine requirement and raised the minimal inhibitory concentration of L-valine, whereas the L-leucine requirement was unaffected. Direct uptake assays showed that YhjE mediates L-isoleucine and L-valine import. Kinetic analysis yielded apparent K_M values of 70.5 μM (L-isoleucine) and 86.7 μM (L-valine), establishing YhjE as a lower-affinity system compared with LIV-I (encoded by the <i>livKHMGF</i> operon and <i>livJ</i> gene) and BrnQ. Energetically, transport was proton-motive-force-dependent: the protonophore carbonyl cyanide <i>m</i>-chlorophenyl hydrazone strongly inhibited uptake, and L-isoleucine addition elicited proton influx as observed by pH measurements. Analysis of transcriptional regulation revealed strict dependence of <i>yhjE</i> promoter activity on Lrp and repression by L-leucine, L-methionine, and L-isoleucine, indicating nutrient-responsive control. Hydroxylamine mutagenesis yielded <i>yhjE</i> variants that permitted growth under L-leucine limitation; across tested alleles, L-leucine uptake increased while L-isoleucine uptake decreased, demonstrating reprogramming of substrate preference via discrete substitutions. Finally, a <i>ΔlivKHMGF ΔbrnQ ΔyhjE</i> triple mutant showed no detectable uptake of L-isoleucine and L-valine at substrate concentrations up to 100 μM, implying that if other routes exist, their contribution at physiological substrate levels is negligible.