Role of Calcium in Assembly of the Azotobacter vinelandii Surface Array

Azotobacter vinelandii grown in a defined medium lacking calcium did not exhibit the tetragonally-arranged surface layer present on calcium-sufficient cells, although each cell type possessed equivalent amounts of surface-localized S-protein. The addition of Ca2+ or Sr2+ to calcium-limited cells suspended in buffer resulted in formation of the S-layer, whereas a similar addition of Mg2+ or Be2+ did not. Incubation of cells with 35SO2- 4 during Ca2+-mediated in vivo reassembly of the S-layer confirmed that the array was formed from previously synthesized, surface-localized S-protein. Rate-zonal sedimentation of S-protein extracted from calcium-limited cells demonstrated tetrameric S-protein subunits characteristic of the native array. S-protein on the surface of calcium-limited cells was not more susceptible to iodination or proteolytic degradation than that on calcium-sufficient cells. These data suggested minimal alteration of the surface layer beyond disorganization of the S-protein subunits. Calcium limitation caused only a minor perturbation of the outer membrane and did not prevent the outer membrane from serving as a template for the in vitro reassembly of externally supplied S-protein subunits. Notably, Mg2+ or Ca2+ mediated in vitro reassembly of the S-layer and produced a layer that was more loosely attached than the native array. These data support the hypothesis that calcium is specifically required for the in vivo assembly of S-protein subunits into a tetragonal surface array.