Research ResourcePhosphoproteomics

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

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Sci. Signal.  15 Sep 2015:
Vol. 8, Issue 394, pp. rs10
DOI: 10.1126/scisignal.aaa3117

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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.

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