Identification and characterization of the S-layer formed on the sheath of Thiothrix nivea.

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.

Elongation pattern and fine structure of the sheaths formed by Thiothrix nivea and Thiothrix fructosivorans.

Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-ß-d-GlcN-(1→4)-ß-d-Glc-(1→]n modified with unusual deoxy sugars. In an attempt to elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were determined in this study. Prior to analysis, both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addition, the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chemically fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.

Presence of N-L-lactyl-D-perosamine residue in the sheath-forming polysaccharide of Thiothrix fructosivorans.

Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1→4)-Glc-(1→]n with side chains of Rha4N-(1→3)-Fuc(1→ 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.