Assessment of Bacterial N-Degron Proteolysis by a Dual-Fluorescence Reporter

Many organisms possess N-degron pathways that link the proteolytic stability of individual proteins to the identity of their N-terminal amino acid. The set of N-degrons recognized by proteolytic machinery, and the regulatory paradigms leading to N-degron exposure, have been well studied in Escherichia coli and several other bacterial species. However, N-degron landscapes remain unexplored in the majority of bacterial phyla, and the extent to which these pathways vary among clades remains unknown. To characterize N-degron proteolysis in Mycolicibacterium smegmatis, a member of Actinomycetota and a model system for pathogenic mycobacteria, we devised a cell-based assay incorporating a dual-fluorescence reporter. We evaluated the proteolysis of mCherry substrates with defined N-terminal sequences by comparing fluorescence levels to a stable GFP reference in plate reader and flow cytometry contexts. By examining proteolysis patterns in wild-type M. smegmatis and cells lacking the N-recognin ClpS, we established that canonical primary destabilizing residues (Leu, Phe, Tyr, Trp) are subject to ClpS-dependent N-degron proteolysis. This dual-fluorescence approach is broadly applicable to interrogation of N-degron pathways across bacterial species.