Isolation and in vitro assembly of the components of the outer S layer of Lampropedia hyalina.
The outermost component of the S layer of Lampropedia hyalina, the punctate layer, is assembled onto an inner perforate layer. The punctate layer is composed of long, tapered cylindrical units centered on p6 symmetry axes and connected by six fine linking arms, joining at the axis of threefold symmetry to create a hexagonal layer with a lattice constant of 25.6 +/- 0.5 nm (J. A. Chapman, R. G. E. Murray, and M. R. J. Salton, Proc. R. Soc. London Ser. B 158:498-513, 1963; R. G. E. Murray, Can. J. Microbiol. 9:593-600, 1963). Extraction of cell envelopes with 100 mM Tris buffer (pH 8) containing 2% deoxycholate resulted in the release of several proteins, but left the S layers intact. The punctate layer was then extracted with 3 M guanidine hydrochloride or 6 M urea, leaving the perforate layer intact. This treatment led to the release of three polypeptides with molecular weights of 60,000, 66,000, and 240,000 (60K, 66K, and 240K polypeptides). These three polypeptides reassembled on the perforate layer as a template to form the S-layer complex or self-assembled to form the punctate layer alone after dialysis of the extract against 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.5) containing 10 mM CaCl2. The self-assemblies were composed of a 240K polypeptide and a 60K polypeptide. The 240K and 60K polypeptides were separated by column chromatography and examined by electron microscopy. The 240K polypeptide appeared in negative stain as a long, flexible structure and assembled into loose arrays with sixfold symmetry with obvious Y-shaped linking elements, while fractions containing both the 60K and 240K polypeptides showed assemblies closely resembling the punctuate layer. Immunoelectron microscopy was used to confirm the presence of both the 60K and 240K polypeptides as components of the punctuate layer.
Austin
J W
JW
Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
Murray
R G
RG
D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure; D002473:Cell Wall / Q000187:drug effects / Q000648:ultrastructure; D003902:Detergents / Q000494:pharmacology; D005051:Eubacterium / Q000648:ultrastructure*; D007106:Immune Sera; D007202:Indicators and Reagents; D008854:Microscopy, Electron
D003902:Detergents; D007106:Immune Sera; D007202:Indicators and Reagents