The first biantennary bacterial secondary cell wall polymer and its influence on S-layer glycoprotein assembly

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-D-GlcpNAc-(1 --> 3)-beta-D-ManpNAc-(1 --> 4)-beta-D-GalpNAc-(1 --> 3)-alpha-D- GlcpNAc-(1 --> 3)-beta-D-ManpNAc-(1 --> 4)-[alpha-D-GalpNAc-(1 --> 3)-alpha-D-GlcpNAc-(1 --> 4)-beta-D-GlcpNAc-(1 --> 3)-beta-D-ManpNAc-(1 --> 4)-beta-D-GalpNAc-(1 --> 3)-alpha-D-GlcpNAc(1 --> 3)-beta-D-ManpNAc- (1 --> 4)-beta-D-GalpNAc - (1 --> 3)-alpha- D-GlcpNAc- (1 --> 3)]-alpha-D-ManpNAc-(1 --> 3)-alpha-D - GlcpNAc-(1 --> 3)-beta-D-ManpNAc-(1 --> 3)-alpha-D-GlcpNAc-(1 --> 3)-alpha-D-GlcpNAc-(1 --> O)-PO2--O-PO2--(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 Algamma-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.