Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Aggregability of ß(1→4)-linked glucosaminoglucan originating from a sulfur-oxidizing bacterium Thiothrix nivea.

ß-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates.