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

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Abstract
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 α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→4)-[α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→4)-β-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→3)]-β-d-ManpNAc-(1→3)-α-d-GlcpNAc-(1→3)-β-d-ManpNAc-(1→3)-α-d-GlcpNAc-(1→3)-α-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 A1γ-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.