S layer regeneration in Methanococcus voltae protoplasts

Abstract

The regeneration of the paracrystalline proteinaceous cell wall (S layer) of the methanogen Methanococcus voltae was examined by electron microscopy. The S layer was removed from the cell surface by exposing the cells to a protoplasting buffer but was completely regenerated by 60-80 min resuspension in a regeneration medium by some repair mechanism (normal cell generation time is 16 h). The protoplast surface appeared in freeze-fracture to be initially smooth and featureless and remained so for the first 20 min in regeneration medium. Thereafter, nascent S layer appeared on the surface in the form of paracrystalline patches of various sizes that were distributed over the entire surface. The patches were closely associated with disturbances of the cell surface curvature which may have been caused by electrostatic interactions. The steps leading to the complete regeneration of the S layer could not be followed due to the sampling times used, but presumably the patches grew large enough to contact neighbouring patches and somehow fused into a coherent layer. Other ultrastructural changes occurred to the cell envelope during S layer regeneration, most notably the recurrence of the intramembrane particles of the concave fracture face of the plasma membrane. Flagella remained attached to the protoplasts and were apparently still functional. The major changes to the protein profile of untreated cells, protoplasts and regenerating protoplasts included the loss and resynthesis of polypeptides at the 76 kDa (S layer polypeptide) and 60 kDa (unknown polypeptide) positions. SDS-PAGE evidence indicated that new S layer polypeptides were synthesized 20 min prior to their appearance as patches on the cell surface. The S layer polypeptide may have been shed from the surface during the early stages of regeneration to form the extracellular sheet-like material observed in large quantity in the regeneration medium. The response of the cell to changes that occur at the cell surface indicates that some feedback mechanism gears the synthesis of S layer and associated envelope components.