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...

Two species belonging to a novel genus of archaea, designated Picrophilus oshimae and Picrophilus torridus, have been isolated from two different solfataric locations in northern Japan. One habitat harboring both organisms was a dry, extremely acidic soil (pH < 0.5) that was heated by solfataric gases to about 55؇C. In the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65؇C; they grew optimally at 60؇C. The pH optimum was 0.7, but growth occurred even around pH 0. Under optimal conditions, the generation time was about 6 h, yielding densities of up to 10 10 cells per ml. The cells were surrounded by a highly filigreed regular tetragonal S-layer, and the core lipids of the membrane were mainly bis-phytanyltetraethers. The 16S rRNA sequences of the two species were about 3% different. The complete 16S rRNA sequence of P. oshimae was 9.3% different from that of the closest relative, Thermoplasma acidophilum. The morphology and physiological properties of the two species characterize Picrophilus as a a novel genus that is a member of a novel family within the order Thermoplasmales.Thermoacidophilic microorganisms thrive on coal refuse piles and in the upper oxygenic zones of acidic geothermally heated waters in solfataras. Sulfuric acid arises in these habitats from the oxidation of H 2 S, either spontaneously or by the action of sulfur-oxidizing bacteria (1). Except for some heterotrophic species of the bacterial genus Bacillus, all organisms isolated from acidic environments with temperatures above 50ЊC have been archaea of the orders Sulfolobales and Thermoplasmales (8, 9). Members of the Sulfolobales are extreme thermophiles that grow optimally at between 75 and 85ЊC. With one exception, they are obligate or facultative heterotrophic aerobes and/or chemolithoautotrophs; in the latter case they are capable of gaining energy by oxidation of sulfur compounds to sulfuric acid or by reduction of sulfur to H 2 S (20). The first described representative of the order Thermoplasmales, Thermoplasma acidophilum, was isolated by Darland et al. (2) from a coal refuse pile. It is unique among the archaea in being devoid of a cell envelope. T. acidophilum is a facultatively anaerobic heterotroph that requires complex organic extracts for growth. Under anerobic conditions, it grows by sulfur respiration (19,21). T. acidophilum grows optimally at 59ЊC and was the prokaryotic record-holder in acidophily, with a pH optimum of around 1.8 to 2 and the ability to still, although barely, grow at around pH 0.4 (1, 2). T. acidophilum and related acidophiles with similar morphologies and physiological properties that have been combined in the taxon Thermoplasma volcanium were later found in various natural habitats around the world (11, 18).Here we report on the isolation and characterization of members of two species of a novel genus and family of thermoacidophilic archaea, which have been obtained by sampling solfataric environments in north...

HslVU is a new two-component protease in Escherichia coli composed of the proteasome-related peptidase HslIV and the ATPase HsIU. We have used electron microscopy and image analysis to examine the structural organization of HslV and HslU homo-oligomers and the active HslVU enzyme. Electron micrographs of HslV reveal ring-shaped particles, and averaging of top views reveal six-fold rotational symmetry, in contrast to other beta-type proteasome subunits, which form rings with seven-fold symmetry. Side views of HslV show two rings stacked together, thus, HslV behaves as dodecamer. The ATPase HslU forms ring-shaped particles in the presence of ATP, AMP-PNP or ADP, suggesting that nucleotide binding, but not hydrolysis, is required for oligomerization. Subunit crosslinking, STEM mass estimation, and analysis of HslU top views indicate that HslU exists both as hexameric and heptameric rings. With AMP-PNP present, maximal proteolytic activity is observed with a molar ratio of HslU to HslV subunits of 1:1, and negative staining electron microscopy shows that HslV and HsIU form cylindrical four-ring structures in which the HsIV dodecamer is flanked at each end by a HslU ring.

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The three-dimensional structure of the regular surface protein (p4 symmetry, lattice constant a = b = 10.5 nm) of Comamonas acidovorans has been determined to a resolution of about 1.5 nm by means of electron microscopy and image processing. Three-dimensional reconstructions were performed using native outer membranes and artificial two-dimensional crystals of the surface protein, which was selectively solubilized by deoxycholate and recrystallized on carbon films. The two-fold symmetric morphological complex is composed of two identical monomers which are in tight contact with the outer membrane and presumably anchored to it by a small hydrophobic domain.