THE ORGANIZATION OF THE PARACRYSTALLINE MULTILAYERED SPACER-PLUGS OF METHANOSPIRILLUM-HUNGATEI

Cells of the filamentous sheathed archeon Methanospirillum hungatei synthesize unusual extracellular multilayered disk-shaped partitions (i.e., plugs) which consist of two types of complementary paracrystalline layers (i.e., a particulate layer consisting of compact elements with massive centers and a holey layer consisting of an open network of pores) with hexagonal symmetry and identical lattice spacing (18.1 nm). The purpose of this study was to determine the three-dimensional architecture of the plug using correlative evidence from thin sectioning, negative staining and image processing, and platinum-shadowing techniques. Heating intact filaments (56 degrees C) suspended in 50 mM carbonate-bicarbonate buffer (pH 9.6) for 30 min resulted in the separation and shedding of the outermost layers from the filament, whereas the innermost layers remained in place. The shed layers were recovered by centrifugation and were identified as holey-type layers. Well-preserved whole plugs extracted using a novel freeze/crush/thaw method were dissociated in 50 mM phosphate buffer (pH 7.4) using mild heat (56 degrees C). These plugs, once platinum-coated and viewed in relief, were found to consist of four paracrystalline layers each with its own amorphous layer. Two particulate layers are located on the cell-facing side and enclose a compartment containing amorphous material. The outside surface of the compartment was covered with a close-fitting holey layer which was attached by an intervening amorphous layer. This amorphous layer was found to bear a faint periodic ''imprint'' with a 18-nm repeat which may be due to the accumulation of transported material. The massive centers of the particulate layer were in exact register with the openings of the holey layers to produce a closed surface. An extra, seemingly redundant, holey layer was found loosely attached to the plug and covered on its outer face with a roughly textured amorphous coating. The various amorphous coatings seem to be necessary for maintaining the plug as a cohesive assembly and for attachment to the cell. (C) 1994 Academic Press, Inc.