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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...

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The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg 2؉ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VAT⌬N complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.

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