Domain structure of the Acetogenium kivui surface layer revealed by electron crystallography and sequence analysis

Abstract

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...

Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

“…The carbohydrate is attached to the protein via 0-glycosidic rather than N-glycosidic bonds (see Results). Recent data indicate that, in bacterial proteins, 0-glycosylation occurs at tyrosin residues, but a consensus sequence around this glycosylated residue has not yet been identified [55,561. The sites at which carbohydrate is attached to the a-amylase are not known, but must lie N-terminal to D466 or C-terminal to approximately residue 1200 (see Results).…”

Expression, secretion and antigenic variation of bacterial S‐layer proteins

“…Except for S-proteins of lactobacilli (pI > 9.5) and Methanothermus (pI = 8.4), all other S-proteins of which the primary structure is known have an acidic isoelectric point. Multiple copies of an Slayer homology (SLH (Lupas et al, 1994)) domain have been found in either the N-or C-terminal part of several S-proteins. This SLH domain is thought to be involved in interaction with either the cell wall or extracellular proteins (see below).…”

Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

“…Interestingly, mutations in the BAC clone that confer reduced but detectable levels of hemolytic activity also were isolated. Insertions producing this activity were in one of two ORFs: an ORF homologous to B. subtilis cell wall amidases (27) or an ORF encoding an S layer homology motif (28,29). Restriction enzyme digestion analysis (not shown) indicated that the two loci were linked closely on BACB61, suggesting a possible functional or transcriptional linkage.…”