The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaBi, flaB2, andflaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaBi, flaB2, and flaB3 flageilins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between thefiaB2 andflaB3 genes appears to increase the resistance of theflaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. TheflaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the M, values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flageilar filaments. Each flageilin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11-or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium.
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.
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