Presence of alternating glucosaminoglucan in the sheath of Thiothrix nivea

Abstract

A sheath-forming sulfa oxidizer, Thiothrix nivea, was mixotrophically cultured in a medium supplemented with acetic acid and sodium disulfide. Its sheath, a microtube-like extracellular supermolecule, was prepared by selectively removing the cells with lysozyme, sodium dodecyl sulfate, and sodium hydroxide. The sheath was not visibly affected by hydrazine treatment, suggesting that it is not a proteinous supermolecule. From the acid hydrolysate of the sheath, glucose and glucosamine were detected in an approximate molar ratio of 1:1. Three other saccharic compounds were detected and recovered by HPLC as fluorescent derivatives prepared by reaction with 4-aminobenzoic acid ethyl ester. Nuclear magnetic resonance (NMR) analysis suggested that one of the derivatives was derived from an unidentified deoxypentose. NMR analysis for the other 2 derivatives showed that they were derived from β-1,4-linked disaccharides and tetrasaccharides, which were composed of glucose and glucosamine. The sheath was readily broken down by weak HCl treatment, releasing an unidentified deoxypentose and polymer. Chemical analysis showed the presence of β-1,4-linked D-Glcp and D-GlcNp in the polymer. NMR analysis revealed that the polymer had a repeating unit of →4)-D-Glcp-(β1→4)-D-GlcNp-(β1→. The solid-state 1D-(13)C NMR spectrum of the polymer in N-acetylated form supported this result. The molecular weight of the polymer was estimated to be 8.2×10(4) by size exclusion chromatography. Based on these results, the sheath of T. nivea is hypothesized to be assembled from alternately β-1,4-linked glucosaminoglucan grafted with unidentified deoxypentose.