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

After treating Mycoplasma pneumoniae cells with the nonionic detergent Trit on X-100, an undefined, structured protein complex remains that is called t he 'Triton X-100 insoluble fraction' or 'Triton shell'. By analogy with euk aryotic cells and supported by ultrastrudural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like stru cture. In this study, the composition of the Triton X-100 insoluble fractio n was defined by electron microscopic screening for possible structural ele ments. and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present. Silver staining of 2-D gels revealed about 100 protei n spots. By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and pa rtial sequence of tryptic peptides of individual proteins. The identified p roteins 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 show n 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. Qua ntitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits alpha and beta of the pyruvate dehydrogen ase complex (PdhA, PdhB), but definite conclusions regarding the compositio n of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry.