Spatial arrangement of gene products of the P1 operon in the membrane of Mycoplasma pneumoniae

Abstract

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 non-membrane-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 Å (1 Å = 0.1 nm) on the tip structure of M. pneumoniae.