Spatial Arrangement of Gene Products of the P1 Operon in the Membrane of Mycoplasma Pneumoniae

The spatial arrangement of three P1 operon-encoded proteins--attachment protein P1 (ORF5 gene product) and the ORF6-derived proteins of 40 and 90 kDa--in the membrane of Mycoplasma pneumoniae M129 was investigated by nearest-neighbor analysis. For these studies, the homobivalent, thiol-cleavable, and nonmembrane-permeating cross-linking reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used. The cross-linked proteins were isolated by immunoprecipitation with antibodies directed against fusion proteins of selected regions of the 40-kDa, the 90-kDa, or the P1 protein. The individual components of the immunoprecipitated protein complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and immunoblot analysis. This study showed that the P1 protein, the ORF6 gene product, and an unidentified 30-kDa protein were linked to each other in the intact bacterial membrane by the reagent DTSSP, indicating that these proteins are located at a maximal distance of 12 A (1 A = 0.1 nm) on the tip structure of M. pneumoniae.