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Recently, controllable, targeted proteolysis has emerged as one of the most promising new strategies to study essential genes and otherwise toxic mutations. One of the principal limitations preventing the wider adoption of this approach is due to the lack of easily identifiable species-specific degrons that can be used to trigger the degradation of target proteins. Here, we report new advancements in the targeted proteolysis concept by creating the first prokaryotic N-terminal targeted proteolysis system. We demonstrate how proteins from the LexA-like protein superfamily can be exploited as species-specific reservoirs of N- and/or C-degrons, which are easily identifiable due to their proximity to strictly conserved residues found among LexA-like proteins. Using the LexA-like regulator HdiR of  Streptococcus mutans , we identified two separate N-degrons derived from HdiR that confer highly efficient constitutive proteolysis upon target proteins when added as N-terminal peptide tags. Both degrons mediate degradation via AAA+ family housekeeping proteases with one degron primarily targeting FtsH and the other targeting the ClpP-dependent proteases. To modulate degron activity, our approach incorporates a hybrid N-terminal protein tag consisting of the ubiquitin-like protein NEDD8 fused to an HdiR degron. The NEDD8 fusion inhibits degron function until the NEDD8-specific endopeptidase NEDP1 is heterologously expressed to expose the N-degron. By fusing the NEDD8-degron tag onto GFP, luciferase, and the pleiotropic regulator RNase J2, we demonstrate that the N-terminal proteolysis approach exhibits far superior performance compared to the classic transcriptional depletion approach and is similarly applicable for the study of highly toxic mutations.

Identification of New Degrons in Streptococcus mutans Reveals a Novel Strategy for Engineering Targeted, Controllable Proteolysis