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Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation

Improved enrichment of phosphorylated proteins from bacteria enables the identification of phosphorylation motifs not found in eukaryotes. Identifying bacterial phosphorylation motifs Mass spectrometry–based analysis of phosphorylated proteins in eukaryotic cells has provided valuable insights into signaling pathways; however, a similar analysis of signaling in bacteria has been hampered by difficulties in purifying sufficient amounts of phosphorylated proteins. Through a refined precipitation step, Lin et al. enhanced the enrichment of phosphorylated proteins from three distinct bacterial strains, which enabled the identification of bacterial-specific phosphorylation motifs by mass spectrometry. An increased understanding of bacterial phosphorylation events may aid in the development of specific antibacterial therapies that are distinct from antibiotics. Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies.