The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacilus sphaericus and Thermus thermophilus, Ompa from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.Acetogenium kivui (19) is a hydrogen-oxidizing, acetogenic bacterium (18) that is moderately thermophilic and grows optimally at 66°C. In spite of its gram-negative staining behavior, its cell wall has gram-positive characteristics. Like many other bacteria, gram positive as well as gram negative, it is covered by a regularly arrayed surface layer (S-layer). This layer has a hexagonal structure and consists of a single 80-kDa protein whose gene has been cloned and sequenced (21). The S-layer protein is modified at four tyrosine residues by long glycan chains that are composed of glucose, galactosamine, and an as-yet-unidentified sugar-related component (22) 8578-2652. Fax: (089) 8578-2641. for S-layer homology) is conserved in several other proteins and discuss possible implications for its function. MATERUILS AND METHODSBacterial strain and growth conditions. A. kivui was obtained from the German collection of Microorganisms (DSM 2030), Braunschweig, Germany. Cells were grown anaerobically in the medium described by Leigh et al. (18), buffered with 50 mM phosphate (pH 6.5) and supplemented with yeast extract (2.0 g per liter), tryptone (2.0 g per liter), and glucose (5.0 g per liter). The growth temperature was between 60 and 640C.S-layer preparation. Cells were harvested in the logarithmic growth phase by centrifugation at 4,500 x g and washed once in distilled water. The peptidoglycan was digested by adding 10 to 20 mg of lysozyme to 100-ml aliquots of cell suspension and incubating the mixture for 6 to 8 h at room temperature. The tilt series chosen for processing comprised 14 projections each. The actual tilt angles ranged from -0.3 to 78.30 (UA) and from 2.3 to 80.9°(PTA). No significant radiation damage was accumulated while recording the tilt series, as the power spectra of nominal 00 tilts...
The regular surface protein structure (S-layer) of Caulobacter crescentus was analyzed by electron microscopy and three-dimensional image reconstruction to a resolution of 2 nm. Projections showed that the S-layer is an array of ring structures, each composed of six subunits that are arranged on a lattice with p6 symmetry. Three-dimensional reconstructions showed that the ring subunits were approximately rod-shaped structures and were perpendicular to the plane of the array, with a linker arm emanating from approximately the middle of the rod, accounting for the connections between the rings. The calculated subunit mass was ca. 100 kDa, very close to the size of RsaA (the protein known to be at least the predominant species in the S-layer) predicted from the DNA sequence of the rsaA4 gene. The core region of the rings creates an open pore 2.5 to 3.5 nm in diameter. The size of the gaps between the neighboring unit cells is in the same range, suggesting a uniform porosity predicted to exclude molecules larger than ca. 17 kDa. Attempts to remove membrane material from S-layer preparations with detergents revealed that the structure spontaneously rearranged into a mirror-image double layer. Negative-stain and thin-section electron microscopy examination of colonies of C. crescentus strains with a mutation in a surface molecule involved in the attachment of the S-layer showed that shed RsaA protein organized into large sheets. The sheets in turn organized into stacks that tended to accumulate near the upper surface of the colony. Image reconstruction indicated that these sheets were also precise mirror-image double layers, and thickness measurements obtained from thin sections were consistent with this finding. The sheets were absent when these mutant strains were grown without calcium, supporting other data that calcium is involved in attachment of the S-layer to a surface molecule and perhaps in subunit-subunit interactions. We propose that when the membrane is removed from S-layer fragments by detergents or the attachment-related surface molecule is absent, the attachment sites of the S-layer align precisely to form a double layer via a calcium interaction.
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