ABSTRACT
Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [â4)-GlcN-(1â4)-Glc-(1â]n with side chains of Rha4N-(1â3)-Fuc(1â at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N-substitution. The sheath was hydrolyzed with concentrated HCl at 0°C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N-lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l-isomer by chiral HPLC analysis. To estimate the N-lactylation degree, the sheath was N-acetylated. N-Acetyl-Rha4N-Fuc-ABEE and N-lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40°C, GlcNAc-ABEE was obtained. For estimation of the N-acetylation degree, the sheath was N-acetylated with deuterated acetic anhydride and then N-acetyl-GlcN-ABEE was prepared. The content of deuterated N-acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D-(1)H NMR spectrum. It was concluded that Rha4N is mostly N-l-lactylated and GlcN is substoichiometrically N-acetylated.
Subject(s)
Mannose/analogs & derivatives , Polysaccharides, Bacterial/chemistry , Thiothrix/metabolism , Chromatography, High Pressure Liquid , Lactic Acid/chemistry , Magnetic Resonance Spectroscopy , Mannose/chemistry , Spectroscopy, Fourier Transform Infrared , Thiothrix/cytology , Thiothrix/ultrastructureABSTRACT
A sheath-forming and sulfur-oxidizing bacterium, Thiothrix fructosivorans, was heterotrophically cultured. The sheath, which is an extracellular microtube, was prepared by selectively removing the cells using lysozyme, sodium dodecyl sulfate, and sodium hydroxide. Solid-state (13)C-nuclear magnetic resonance (NMR) spectrum revealed that the sheath is assembled from a glycan possessing acetyl and methyl groups. When the sheath was deacetylated, the original microtube structure was lost and the sheath became soluble under acidic conditions, revealing the importance of acetyl groups in maintaining the sheath structure. Equimolar d-glucose, d-glucosamine, and l-fucose were detected in the acid hydrolysate of the sheath by gas liquid chromatography. In addition to these sugars, ß-GlcN-(1â4)-Glc and unidentified sugar were detected by analyzing the hydrolysate using high-performance liquid chromatography analysis. (1)H and (13)C NMR spectroscopy was used to identify a disaccharide composed of 4-deoxy-4-aminorhamnose (perosamine, Rha4N) and fucose. N-Acetyl-perosamine prepared from the disaccharide was polarimetric and exhibited a d-configuration. The previously unidentified disaccharide was found to be α-d-Rhap4N-(1â3)-d-Fuc. According to (1)H and (13)C NMR analyses, the deacetylated sheath-forming polysaccharide was found to h have a main chain of [â4)-ß-d-GlcpN-(1â4)-ß-d-Glcp-(1â]n, to which disaccharide side chains of α-d-Rhap4N-(1â3)-α-l-Fucp-(1â were attached at position 3 of Glc.