Illuminating protein interactions: Photo-Leucine crosslinking in Bacillus subtilis

Understanding the intricate network of protein-protein interactions (PPIs) is essential for unraveling the complexities of cellular function. Photo-crosslinking has emerged as an exciting new method for probing PPIs in many organisms but has not yet found widespread application in bacteria. In this study, we aim to apply photo-crosslinking with photo-leucine in the well-studied gram-positive model organism Bacillus subtilis, laying the groundwork for its potential future applications.

We show that photo-leucine becomes highly toxic for B. subtilis and investigated the mechanisms of photo-leucine uptake. We provide evidence that the branched-chain amino acid importers BcaP and BraB are involved in the uptake of photo-leucine. Additionally, we unveil the pivotal role of the bipartite exporter AzlCD in facilitating the efficient export of photo-leucine from the cell. Furthermore, our investigation uncovers a previously uncharacterized exporter, AexB, belonging to the novel "sleeping beauty" group of the EamA family [1]. Notably, AexB is found to be positively regulated by the transcription factor AerB, a member of the AraC family. Additionally, we isolated a mutant highly resistant to photo-leucine and identified a single amino acid exchange in the leucine-tRNA synthetase LeuS responsible for this resistance. Using a molecular docking approach, we show that the mutation alters the binding cavity for leucine in a manner that only allows leucine, but not photo-leucine, to bind productively. Furthermore, incorporation rate studies in the suppressor mutant with the LeuS mutation revealed that the photo-leucine incorporation rate was below the detection limit. This work presents the homeostasis of photo-leucine in B. subtilis, laying the groundwork for future application of photo-crosslinking and thus the possible identification of novel protein-protein interactions as well as structure refinement.

[1] Warneke et al. (2023) mBio 15:e03456-23.