Photo-leucine incorporation in Bacillus subtilis : Identification of transporters and of a selectivity determinant in the Leu-tRNA synthetase

The elucidation of the intricate network of proteins is essential for our understanding of cellular function. In vivo crosslinking is a powerful tool for probing these interactions, with photoactivatable amino acids representing a promising next step. Here, we evaluated the application of photo-leucine in the gram-positive model organism Bacillus subtilis . This amino acid analog is incorporated into proteins, but high concentrations inhibit the growth of B. subtilis . We investigated photo-leucine homeostasis and identified the branched-chain amino acid importers BcaP and BraB as well as the bipartite exporter AzlCD as key players of its uptake and export, respectively. Additionally, we identified a previously uncharacterized exporter, AexB, a member of the "sleeping beauty" group of EamA amino acid exporters. Expression of the aexB gene is positively regulated by the transcription factor AerB, that is normally normally inactive. Selective pressure by photo-leucine triggered the acqusition of a point mutation in aerB , allowing AerB to activate aexB expression. Mutants highly resistant to photo-leucine carried a single amino acid exchange, Ala-494 to Thr, in the leucine-tRNA synthetase LeuS. Molecular docking analysis revealed that this mutation alters the leucine-binding pocket, in a manner that still allowed binding of leucine, but not photo-leucine. While high photo-leucine incorporation is beneficial for effective crosslinking, our results suggest that B. subtilis intrinsically limits its incorporation to maintain cellular tolerance. Thus, evolutionary processes provide adaptive mechanisms to adjust amino acid analogue incorporation to tolerable levels, making photo amino acids a candidate for life cell investigation of protein struture and interactions.