AbstractThe diversity of non-canonical amino acids (ncAAs) endows proteins with new features for a variety of biological studies and biotechnological applications. The genetic code expansion strategy, which co-translationally incorporates ncAAs into specific sites of target proteins, has been applied in many organisms. However, there have been only few studies on pathogens using genetic code expansion. Here, we introduce this technique into the human pathogen Salmonella by incorporating p-azido-phenylalanine, benzoyl-phenylalanine, acetyl-lysine, and phosphoserine into selected Salmonella proteins including a microcompartment shell protein (PduA), a type III secretion effector protein (SteA), and a metabolic enzyme (malate dehydrogenase), and demonstrate practical applications of genetic code expansion in protein labeling, photocrosslinking, and post-translational modification studies in Salmonella. This work will provide powerful tools for a wide range of studies on Salmonella.