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.

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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.