Recent investigations have shown that a number of crystalline surface layers (S-layers) (for reviews, see references 4 and 19) of members of the family Bacillaceae are composed of glycosylated S-layer protein protomers (18,19). Most of the eubacterial S-layer glycoprotein glycans investigated contain polysaccharide regions composed of oligosaccharide repeating units, similar to the O antigens of gram-negative bacteria (20). Considerable diversity exists among the repeating-unit structures, even among closely related strains within a species (17,22).The O-antigen-like repeats of the S-layer glycan from Thermoanaerobacter (Clostridium) thermohydrosulfuricus L111-69 have the structurewhich in turn is linked to the S-layer polypeptide by an Oglycosidic linkage to tyrosine (5).Tyrosine was found not only in all pronase-derived glycopeptides from the investigated T. thermohydrosulfuricus strains (17, 22) but, unexpectedly, also in the glycopeptides analogously obtained from S-layer glycoproteins of Bacillus alvei CCM 2051. In a previous work, the repeating-unit structure of the respective glycan has been identified asTo compare the tyrosine-linked cores of the S-layer glycoprotein glycans from T. thermohydrosulfuricus and B. alvei, a suitably large amount of a pronase-derived glycopeptide from the B. alvei S-layer glycoprotein was prepared. The low-intensity nuclear magnetic resonance (NMR) signals corresponding to the core sugars were analyzed and compared with the results of Bock et al. (5). Here, we report on the complete S-layer glycan structure of B. alvei CCM 2051 and draw conclusions about similar core structures in tyrosine-linked eubacterial Slayer glycoproteins.
MATERIALS AND METHODSGrowth of bacteria. B. alvei CCM 2051 was obtained from the Czechoslovakian Collection of Microorganisms, Brno, Czech Republic, and was grown at 33ЊC as described previously (1, 21).Analytical methods and electron microscopy. Carbohydrate and amino acid analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electron microscopy, and sequence analysis were performed according to published methods (5). The phosphate contents of the glycopeptide preparations were determined as described elsewhere (15).Isolation of S-layer glycoprotein and S-layer glycopeptides. Following isolation of intact S-layer glycoprotein, glycopeptides were prepared by exhaustive pronase digestion (1). Final purification of the glycopeptide fractions of interest included gel filtration over Bio-Gel P-4 and P-100 columns and cation-exchange chromatography over Dowex 50W-X8 resin, chromatofocusing between pH 9.0 and 5.6, and reversed-phase high-performance liquid chromatography (RP-HPLC) as described previously (5). The required glycopeptides were lyophilized and stored at Ϫ18ЊC. Sequence analysis of the glycopeptides was performed as described previously (5).NMR measurements. Solutions of ϳ40 mg of glycopeptide in 0.5 ml of D 2 O were used. Spectra at 320 K in 5-mm-diameter tubes at 500. C were recorded with a Bruker AMX-500 spectrometer. The carb...
The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed: [(Pyr4,6)-beta-D-ManpNAc-(1-->4)-beta-D-GlcpNAc-(1-->3)]n-11-(Pyr4,6)-beta-D-ManpNAc-(1-->4)-alpha-D-GlcpNAc-(1-->O)-PO2-O-PO2-(O-->6)-MurNAc- Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. 31P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex. In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.
The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain. Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences. Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions. These
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