Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114

Abstract

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatij) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.

Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

“…Flagellin and S-layer Purification-Crude flagellar filament preparations were isolated by shearing followed by banding in a KBr gradient (8). For isolation of flagella containing some attached basal structure (whole intact flagella), cells were extracted with the non-ionic detergent OP-10 without prior flagella shearing as described by Bardy et al (6).…”

“…Microbial Strains and Growth Conditions-M. voltae cells were grown in Balch medium III under a head space gas mixture of 80% H 2 , 20% CO 2 at 37°C as described previously (8).…”

“…Glycosylation of archaeal flagellin using glycoprotein-specific stains such as thymol sulfuric acid or periodic acid-Schiff is commonly reported (7,8), although a detailed structural analysis of the post-translational modification present is limited to a single organism, Halobacterium salinarum (formerly Halobacterium halobium) (9). The flagellin of this organism has been shown to be glycosylated with N-linked sulfated oligosaccharides composed of glucose in 1,4 linkage to hexuronic acids (10,11).…”

Flagellin from an Incompatible Strain of Pseudomonas avenae Induces a Resistance Response in Cultured Rice Cells

“…Immunogold Labeling of Whole Cells and Transmission Electron Microscopy-Cells were suspended in 0.85% (w/v) NaCl and prepared essentially as described previously (27). Briefly, cells were fixed on Formvar-and carbon-coated grids with 2% (v/v) glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4).…”

The flagellar bundle of Halobacterium salinarium is inserted into a distinct polar cap structure

“…Upon the increase of the concentration of cholate in the elution buffer to 1%, flagella but no flagellated patches were seen, suggesting that the latter are sensitive to this detergent. Some of the detached flagellar filaments appeared to be equipped with a knob-like structure at their base similar in shape to the basal body of Methanococcus voltae (6). However, the quality of the preparation was not good enough to definitely assign them to the flagellar basal body.…”