Structure of the Azotobacter vinelandii surface layer.
Electron microscopy of the Azotobacter vinelandii tetragonal surface array, negatively stained with ammonium molybdate in the presence of 1 mM calcium chloride, showed an apparent repeat frequency of 12 to 13 nm. Image processing showed dominant tetrad units alternating with low-contrast cruciform structures formed at the junction of slender linkers extending from corner macromolecules of four adjoining dominant units. The actual unit cell showed p4 symmetry, and a = b = 18.4 nm. Distilled water extraction of the surface array released a multimeric form of the single 60,000 molecular-weight protein (S protein) which constitutes the surface layer. The molecular weight of the multimer was estimated at 255,000 by gel filtration, indicating a tetrameric structure of four identical subunits and suggesting that this multimer was the morphological subunit of the S layer. Tetrameric S protein exhibited low intrinsic stability once released from the outer membrane, dissociating into monomers when incubated in a variety of buffers including those which served as the base for defined media used to cultivate A. vinelandii. The tetramer could not be stabilized in these buffers at any temperature between 4 and 30 degrees C, but the addition of 2 to 5 mM Ca2+ or Mg2+ completely prevented its dissociation into monomers. Circular dichroism measurements indicated that the secondary structure of the tetramer was dominated by aperiodic and beta-sheet conformations, and the addition of Ca2+ did not produce any gross changes in this structure. Only the tetrameric form of S protein was able to reassemble in vitro in the presence of divalent cations onto the surface of cells stripped of their native S layer.
D001395:Azotobacter / Q000254:growth & development / Q000648:ultrastructure*; D001426:Bacterial Proteins / Q000302:isolation & purification; D002122:Calcium Chloride; D002462:Cell Membrane / Q000648:ultrastructure; D002942:Circular Dichroism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron / Q000379:methods; D008970:Molecular Weight; D008982:Molybdenum; D011487:Protein Conformation
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008982:Molybdenum; D002122:Calcium Chloride; C022175:ammonium molybdate