Identification of arginine phosphorylation inMycolicibacterium smegmatis

Tuberculosis is a leading cause of worldwide infectious mortality. The prevalence of multidrug-resistant Mycobacterium tuberculosis ( Mtb ) infections drives an urgent need to exploit new drug targets. One such target is the ATP-dependent protease ClpC1P1P2, which is strictly essential for viability. However, few proteolytic substrates of mycobacterial ClpC1P1P2 have been identified to date. Recent studies in Bacillus subtilis have shown that the orthologous ClpCP protease recognizes proteolytic substrates bearing post-translational arginine phosphorylation. While several lines of evidence suggest that ClpC1P1P2 is similarly capable of recognizing phosphoarginine-bearing proteins, the existence of phosphoarginine modifications in mycobacteria has remained in question. Here, we confirm the presence of post-translational phosphoarginine modifications in Mycolicibacterium smegmatis ( Msm ), a nonpathogenic surrogate of Mtb . Using a phosphopeptide enrichment workflow coupled with shotgun phosphoproteomics, we identify arginine phosphosites on several functionally diverse targets within the Msm proteome. Interestingly, phosphoarginine modifications are not upregulated by heat stress, suggesting divergent roles in mycobacteria and Bacillus . Our findings provide new evidence supporting the existence of phosphoarginine-mediated proteolysis by ClpC1P1P2 in mycobacteria and other actinobacterial species.