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. hungatii]) 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.