Identification of Effectors: Precipitation of Supernatant Material
Bacterial secretion systems allow the transport of proteins, called effectors, as well as external machine components in the extracellular medium or directly into target cells. Comparison of the secretome, i.e. the proteins released in the culture medium, of wild-type and mutant cells provides information on the secretion profile. In addition, mass spectrometry analyses of the culture supernatant of bacteria grown in liquid culture under secreting conditions allows the identification of secretion system substrates. Upon identification of the substrates, the secretion profile serves as a tool to test the functionality of secretion systems. Here we present a classical method used to concentrate the culture supernatant, based on trichloroacetic acid precipitation.
Key wordsSupernatant TCA precipitation Secretome
This work was supported by the Centre National de la Recherche Scientifique, the Aix-Marseille Université and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02 and ANR-15-CE11-0019-01). The doctoral studies of N.F. are supported by the ANR-14-CE14-0006-02 grant.
- 8.Hood RD, Singh P, Hsu F, Güvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL, Li M, Schwarz S, Wang WY, Merz AJ, Goodlett DR, Mougous JD (2010) A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7:25–37CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Veith PD, Chen YY, Gorasia DG, Chen D, Glew MD, O’Brien-Simpson NM, Cecil JD, Holden JA, Reynolds EC (2014) Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors. J Proteome Res 13:2420–2432CrossRefPubMedGoogle Scholar