Advanced tool for characterization of microbial cultures by combining cytomics and proteomics
- 448 Downloads
Flow cytometry approaches are applicable to recover sub-populations of microbial cultures in a purified form. To examine the characteristics of each sorted cell population, Omics technologies can be used for comprehensively monitoring cellular physiology, adaptation reactions, and regulated processes. In this study, we combined flow cytometry and gel-free proteomic analysis to investigate an artificial mixed bacterial culture consisting of Escherichia coli K-12 and Pseudomonas putida KT2440. Therefore, a filter-based device technique and an on-membrane digestion protocol were combined in conjunction with liquid chromatography and mass spectrometry. This combination enabled us to identify 903 proteins from sorted E. coli K-12 and 867 proteins from sorted P. putida KT2440 bacteria from only 5 × 106 cells of each. Comparative proteomic analysis of sorted and non-sorted samples was done to prove that sorting did not significantly influence the bacterial proteome profile. We further investigated the physicochemical properties, namely M r, pI, hydropathicity, and transmembrane helices of the proteins covered. The on-membrane digestion protocol applied did not require conventional detergents or urea, but exhibited similar recovery of all protein classes as established protocols with non-sorted bacterial samples.
KeywordsFilter-based concentration Flow cytometry Microbial cultures Mass spectrometry
We are grateful to Christine Schumann, Michaela Öhler, and Ulrike Lissner for excellent technical assistance. The work in the Interfaculty Institute of Genetics and Functional Genomics (U.V., F.S.) was supported within the framework of the collaborative research project SFBTRR34 by the Deutsche Forschungsgemeinschaft.
- Günther S, Trutnau M, Kleinsteuber S, Hause G, Bley T, Roske I, Harms H, Müller S (2009) Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4′, 6′-diamidino-2-phenylindole) and tetracycline labeling. Appl Environ Microbiol 75:2111–2121CrossRefGoogle Scholar
- Müller S, Nebe-von-Caron G (2010) Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol Rev 34(4):554-87Google Scholar
- Paape D, Lippuner C, Schmid M, Ackermann R, Barrios-Llerena ME, Zimny-Arndt U, Brinkmann V, Arndt B, Pleissner KP, Jungblut PR, Aebischer T (2008) Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes. Mol Cell Proteomics 7:1688–1701CrossRefGoogle Scholar
- Schmidt F, Donahoe S, Hagens K, Mattow J, Schaible UE, Kaufmann SH, Aebersold R, Jungblut PR (2004) Complementary analysis of the Mycobacterium tuberculosis proteome by two-dimensional electrophoresis and isotope-coded affinity tag technology. Mol Cell Proteomics 3:24–42Google Scholar
- Schmidt F, Scharf SS, Hildebrandt P, Burian MS, Bernhardt J, Dhople VM, Kalinka J, Gutjahr M, Hammer E, Völker U (2010) Time resolved quantitative proteome profiling of host—pathogen interactions: the response of S. aureus RN1HG to internalisation by human airway epithelial cells. Proteomics (in press)Google Scholar