Capsular and exopolysaccharides play crucial roles in the biology of many bacteria, acting either as virulence determinants that withstand host-cell defenses, or in establishing symbiotic relationships between bacteria and plants. More than 80 different capsular structures (known as K antigens) are produced by Escherichia coli isolates and these are subdivided into four different groups based on genetic and biochemical criteria (1). Surface polysaccharides with similar features are formed by other bacterial genera. The his-linked cps loci encode enzymes for the assembly of group 1 capsular K-antigens in E. coli and Klebsiella pneumoniae. The cps loci all contain a conserved region comprising the first 4 genes (orfX, wza, wzb, and wzc cps ) (2), indicating a shared role in CPS 1 expression. Following the conserved genes is a serotype-specific region encoding enzymes that participate in synthesis of polysaccharide repeat units and their polymerization via a Wzy-dependent mechanism (3). The Wzy-mediated polymerization reaction is thought to result in formation of an undecaprenyl pyrophosphate-linked glycan at the periplasmic face of the plasma membrane. The nascent polymer is then translocated to the cell surface via a process that requires outer membrane complexes formed by multimers of Wza cps (4). These complexes resemble the "secretins" for secretion of proteins via type II and type III systems.
Several glycan structures of S-layer glycoproteins of gram-positive eubacteria were compared with the principal structural organization of O-antigens of lipopolysaccharides of gram-negative eubacteria. Further, activated intermediates of the biosynthetic pathway of S-layer glycans were compared with activated intermediates of the route of assembly of lipopolysaccharide O-antigens. As a result, at least structural similarities between both types of molecules have been clearly observed. More detailed studies of the assembly of S-layer glycans are required to unambiguously demonstrate the extent to which the biosynthetic pathways of both molecules are related.
The characterization of the S-layer glycoprotein of Bacillus thermoaerophilus revealed unexpected novelties. The isolation and purification procedure had to be changed due to complete solubility in aqueous buffers of the constituting S-layer protomers. Upon degradation of the S-layer glycoprotein by pronase and purification of the products by gel filtration, ion-exchange chromatography, chromatofocusing and HPLC, one representative glycopeptide fraction was selected for further characterization. From the combined evidence of composition analysis, chemical degradation, NMR spectroscopy experiments and comparison with synthesized model substance, we propose the following repeating unit structure of the glycan chain: -->4)-alpha-L-Rhap-(1-->3)-beta-D-glycero-D-manno-Hepp-(1--> This is the first description of heptose residues occurring as a constituent of S-layer glycoproteins of gram-positive eubacteria.
Isolate GS4-97 was purified from an extraction juice sample of an Austrian beet sugar factory and affiliated to the newly described species Aneurinibacillus thermoaerophilus. It is closely related to the type strain of this species, A.thermoaerophilus L420-91(T), and possesses a square surface layer (S-layer) array composed of identical glycoprotein monomers as its outermost cell envelope component. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified S-layer showed an apparent molecular mass of approximately 109,000. After thorough proteolytic degradation of this material by pronase E and purification of the reaction mixture by gel permeation, chromatofocusing, and reversed-phase chromatography, a homogeneous glycopeptide fraction was obtained which was subjected to one- and two-dimensional nuclear magnetic resonance spectroscopy. The combined chemical and spectroscopic evidence, together with N-terminal sequencing, suggest the following structure of the O-glycosidically linked S-layer glycan chain of the glycopeptide: This is the first description of a beta-d-GalNAc-Thr linkage in glycoproteins.
The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.
During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins. As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail. Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer. Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.
The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze-thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer-peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->4)-[alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)]-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->O)-PO(2)(-)-O-PO(2)(-)-(O-->6)-MurNAc- (where MurNAc is N -acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N -acetylmuramic acid residue, in average, of the A1gamma-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S-layer glycoprotein in vitro. Initial crystallization experiments performed with the soluble S-layer glycoprotein revealed that the assembly property could be restored upon dissociation of the polymer by the addition of poly(ethylene glycols). The formed two-dimensional crystalline S-layer self-assembly products exhibited the same lattice symmetry as observed on intact bacterial cells.
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