Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry

Microbiology. 2001 Apr;147(Pt 4):1045-1057. doi: 10.1099/00221287-147-4-1045.


After treating Mycoplasma pneumoniae cells with the nonionic detergent Triton X-100, an undefined, structured protein complex remains that is called the 'Triton X-100 insoluble fraction' or 'Triton shell'. By analogy with eukaryotic cells and supported by ultrastructural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like structure. In this study, the composition of the Triton X-100 insoluble fraction was defined by electron microscopic screening for possible structural elements, and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present. Silver staining of 2-D gels revealed about 100 protein spots. By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and partial sequence of tryptic peptides of individual proteins. The identified proteins belonged to several functional categories, mainly energy metabolism, translation and heat-shock response. In addition, lipoproteins were found and most of the proteins involved in cytadherence that were previously shown to be components of the Triton X-100 insoluble fraction. There were also 11 functionally unassigned proteins. Based on sequence-derived predictions, some of these might be potential candidates for structural components. Quantitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits alpha and beta of the pyruvate dehydrogenase complex (PdhA, PdhB), but definite conclusions regarding the composition of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / analysis
  • Bacterial Proteins / isolation & purification*
  • Blotting, Western
  • Cell Fractionation
  • Cytoskeletal Proteins / analysis
  • Cytoskeletal Proteins / isolation & purification
  • Detergents
  • Electrophoresis, Gel, Two-Dimensional
  • Heat-Shock Proteins / analysis
  • Heat-Shock Proteins / isolation & purification
  • Lipoproteins / analysis
  • Lipoproteins / isolation & purification
  • Mass Spectrometry
  • Microscopy, Electron
  • Mycoplasma pneumoniae / chemistry*
  • Mycoplasma pneumoniae / ultrastructure
  • Octoxynol
  • Peptide Fragments / analysis


  • Bacterial Proteins
  • Cytoskeletal Proteins
  • Detergents
  • Heat-Shock Proteins
  • Lipoproteins
  • Peptide Fragments
  • Octoxynol