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

Cells of the filamentous sheathed archeon Methanospirillum hungatei sy nthesize 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 po res) with hexagonal symmetry and identical lattice spacing (18.1 nm). The purpose of this study was to determine the three-dimensional archi tecture of the plug using correlative evidence from thin sectioning, n egative staining and image processing, and platinum-shadowing techniqu es. Heating intact filaments (56 degrees C) suspended in 50 mM carbona te-bicarbonate buffer (pH 9.6) for 30 min resulted in the separation a nd shedding of the outermost layers from the filament, whereas the inn ermost layers remained in place. The shed layers were recovered by cen trifugation and were identified as holey-type layers. Well-preserved w hole plugs extracted using a novel freeze/crush/thaw method were disso ciated 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 surf ace of the compartment was covered with a close-fitting holey layer wh ich was attached by an intervening amorphous layer. This amorphous lay er was found to bear a faint periodic ''imprint'' with a 18-nm repeat which may be due to the accumulation of transported material. The mass ive centers of the particulate layer were in exact register with the o penings of the holey layers to produce a closed surface. An extra, see mingly redundant, holey layer was found loosely attached to the plug a nd 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) 19 94 Academic Press, Inc.