Chemical characterization of the regularly arranged surface layer glycoprotein of Clostridium thermosaccharolyticum D120-70

Clostridium thermosaccharolyticum D120-70 possesses as its outermost cell envelope layer a square-arranged array of glycoprotein molecules. SDS/polyacrylamide gel electrophoresis of the purified surface layer showed a broadened band in the molecular mass range of about 115 kDa which, upon periodic acid/Schiff staining, gave a positive reaction. After proteolytic degradation of this material, two glycopeptide fractions were obtained. One- and two-dimensional nuclear magnetic resonance studies, together with methylation analysis and periodate oxidation, were used to determine the structures of the polysaccharide portions of these glycopeptides. The combined chemical and spectroscopic evidence suggests the following structures: (formula; see text).     

Charge distribution on the S layer of Bacillus stearothermophilus NRS 1536/3c and importance of charged groups for morphogenesis and function.

The distribution and functional significance of charged groups on the outer and inner faces of the S layer from Bacillus stearothermophilus NRS 1536/3c was investigated. Chemical modification of the exposed amino or carboxyl groups was performed on whole cells, isolated S layers self-assembled in vitro, and cell wall fragments (S layer attached to the peptidoglycan-containing sacculus). Without chemical modification, S layer self-assembly products could be labeled with polycationic ferritin, while S layers on whole cells could not. Following treatment with glutaraldehyde, whole cells were uniformly labeled with polycationic ferritin. Whole cells treated with glutaraldehyde and glycine methyl ester in the presence of carbodiimide did not bind polycationic ferritin significantly above background. Treatment of cell wall fragments with amino-specific, homobifunctional cross-linkers or with carbodiimide alone rendered the S layer protein nonextractable with sodium dodecyl sulfate. After amidation of the accessible carboxyl groups, the modified, guanidine hydrochloride-extractable S layer protomers did not self-assemble into regularly structured lattices. N-Amidination with ethylacetimidate did not interfere with the self-assembly of the isolated protomers. N-Acetylation resulted in a considerable destabilization of the S layer lattice, as seen by the release of a large amount of modified protomers during the reaction. N-Succinylation led to a complete disintegration of the protein lattice. These results indicated that only the inner face of the S layer carried a net negative charge. On both faces, free amino and carboxyl groups of adjacent protomers were arranged in proximity so as to contribute by electrostatic interactions to the cohesion of the protomers in the two-dimensional array. The native charge of the protomers was required for both the in vitro self-assembly of the isolated subunits and the maintenance of the structural integrity of the S layer lattice. Among other functions, the biological significance of the S layers may be in masking the electronegative charge of the cell wall proper.     

Use of regularly structured bacterial cell envelope layers as matrix for the immobilization of macromolecules

Summary Carboxyl groups present on the outer face of the hexagonally ordered S-layer lattices from Bacillus stearothermophilus PV72 and Clostridium thermohydrosulfuricum L111-69 were activated with carbodiimide. The reaction of the activated carboxyl groups with free amino groups of low molecular weight nucleophiles was controlled by labelling with polycationized ferritin, a net positively charged topographical marker for electron microscopy, which densely binds to S-layers possessing free carboxyl groups. Carbodiimide-activated carboxyl groups were also allowed to react with amino groups of ferritin (MW 440 000) and invertase (MW 270 000). Covalent attachment of ferritin was examined by electron microscopy. Using invertase, approximately 1 mg enzyme was bound per mg S-layer protein indicating a high packing density of invertase molecules on the outer face of the S-layer lattice. The immobilized invertase retained 70% of its original activity.     

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains

Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique.During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein.     

Structural and chemical characterization of S-layers of selected strains ofBacillus stearothermophilus andDesulfotomaculum nigrificans

The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each ofBacillus stearothermophilus andDesulfotomaculum nigrificans were compared. Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent. The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices. In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products. The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic. With the exception of the S-layers of two strains ofB. stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated. Among these strains significant differences in the amount and composition of the glycan portions were found. Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.     

Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90°C

A novel type of bacterium has been isolated from various geothermally heated locales on the sea floor. The organisms are strictly anaerobic, rod-shaped, fermentative, extremely thermophilic and grow between 55 and 90°C with an optimum of around 80°C. Cells show a unique sheath-like structure and monotrichous flagellation. By 16S rRNA sequencing they clearly belong to the eubacteria, although no close relationship to any known group could be detected. The majority of their lipids appear to be unique in structure among the eubacteria. Isolate MSB8 is described as Thermotoga maritima, representing the new genus Thermotoga.Dedicated to Otto Kandler on the occasion of his 65th birthday Present addresses: University of South Dakota, Vermillion, USA; University of Illinois, Urbana, USA; Universität für Bodenkultur, Wien, Austria     

Gefrierätzung verschiedener Stämme von Bacillus sphaericus

Zusammenfassung Drei thermophile und ein mesophiler Bacillus sphaericus-Stamm wurden gefriergeätzt und ihre Feinstruktur verglichen. Mit Ausnahme eines thermophilen Stammes konnte bei allen untersuchten Organismen eine aus 13 nm großen Einheiten aufgebaute, rechtwinkelig geordnete Zellwandoberflächenstruktur nachgewiesen werden. Die Zellwand ließ sich bei sämtlichen Stämmen zweischichtig aufspalten. An ihren Querbrüchen traten 10–20 nm dicke Fibrillen auf, die als Subeinheiten der Zellwand gedeutet werden. Das Gefrierätzbild der Cytoplasmamembran läßt eine Deutung im Sinne einer teilweisen Aufspaltung längs einer zentralen Ebene zu.An aufgebrochenen Reservestoffgranula wurden hornartige Artefakte dargestellt, deren Entstehung teilweise auf eine schlagartige Expansion eines komprimierten, streng abgrenzbaren zentralen Teiles zurückgeführt wird.

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Freeze etching of different strains of Bacillus sphaericus

Summary The fine structures of three thermophilic and one mesophilic strain of Bacillus sphaericus have been compared by means of freeze etching. With the exception of one thermophilic strain, all strains had a rectangular cell wall surface structure consisting of 13 nm units. With all strains it was possible to split the cell wall into two layers. On its cross fractures 10–20 nm thick fibrils could be observed, presumably subunits of the cell wall.The image yielded by the freeze etched cytoplasmic membrane can be interpreted as due to partial cleaving along a central plane. The formation of hornlike artefacts rising from cross-fractured storage granules is explained partially by a sudden expansion of a compressed central part.

     

Dynamics in oxygen-induced changes in S-layer protein synthesis from Bacillus stearothermophilus PV72 and the S-layer-deficient variant T5 in continuous culture and studies of the cell wall composition.

Stable synthesis of the hexagonally ordered (p6) S-layer protein from the wild-type strain of Bacillus stearothermophilus PV72 could be achieved in continuous culture on complex medium only under oxygen-limited conditions when glucose was used as the sole carbon source. Depending on the adaptation of the wild-type strain to low oxygen supply, the dynamics in oxygen-induced changes in S-layer protein synthesis was different when the rate of aeration was increased to a level that allowed dissimilation of amino acids. If oxygen supply was increased at the beginning of continuous culture, synthesis of the p6 S-layer protein from the wild-type strain (encoded by the sbsA gene) was immediately stopped and replaced by that of a new type of S-layer protein (encoded by the sbsB gene) which assembled into an oblique (p2) lattice. In cells adapted to a prolonged low oxygen supply, first, low-level p2 S-layer protein synthesis and second, synchronous synthesis of comparable amounts of both types of S-layer proteins could be induced by stepwise increasing the rate of aeration. The time course of changes in S-layer protein synthesis was followed up by immunogold labelling of whole cells. Synthesis of the p2 S-layer protein could also be induced in the p6-deficient variant T5. Hybridization data obtained by applying the radiolabelled N-terminal and C-terminal sbsA fragments and the N-terminal sbsB fragment to the genomic DNA of all the three organisms indicated that changes in S-layer protein synthesis were accompanied by chromosomal rearrangement. Chemical analysis of peptidoglycan-containing sacculi and extraction and recrystallization experiments revealed that at least for the wild-type strain, a cell wall polymer consisting of N-acetylglucosamine and glucose is responsible for binding of the p6 S-layer protein to the rigid cell wall layer.