CHARACTERIZATION OF THE CELL-WALL OF THE SHEATHED METHANOGEN METHANOSPIRILLUM-HUNGATEI GP1 AS AN S-LAYER

The cell wall of Methanospirillum hungatei GP1 is a labile structure t hat has been difficult to isolate and characterize because the cells w hich it encases are contained within a sheath. Cell-sized fragments, 5 60 nm wide by several micrometers long, of cell wall were extracted by a novel method involving the gradual drying of the filaments in 2% (w t/vol) sodium dodecyl sulfate and 10% (wt/vol) sucrose in 50 mM N-2-hy droxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer containin g 10 mM EDTA. The surface was a hexagonal array (a = b = 15.1 nm) poss essing a helical superstructure with a ca. 2.5 degrees pitch angle. In shadowed relief, the smooth outer face was punctuated with deep pits, whereas the inner face was relatively featureless. Computer-based two -dimensional reconstructed views of the negatively stained layer demon strated 4.0- and 2.0-nm-wide electron-dense regions on opposite sides of the layer likely corresponding to the openings of funnel-shaped cha nnels. The face featuring the larger openings best corresponds to the outer face of the layer. The smaller opening was encircled by a stalk- like mass from which 2.2-nm-wide protrusions were resolved. The cell w all in situ was degraded at pH 9.6 at 56 degrees C but was unaffected at pH 7.4 at the same temperature. The cell wall was composed of two n onglycosylated polypeptides (114 and 110 kDa). The cell wall resembled an archaeal S layer and may function in regulating the passage of sma ll (<lO.kDa) sheath precursor proteins (G. Southam and T. J. Beveridge , J. Bacteriol. 174:6460-6470, 1992).