Biochemical and Structural Characterization of an Archaeal Flagella

Abstract

Archaea cells possess a variety of motility and adhesion surface apparatus such as pili, hami, cannulae, and flagella which are genetically and biochemically unique to this domain of life. To explore the identity and properties of flagella from the model methanogenic archaean Methanospirillum hungatei strain JF1, we isolated the polar flagellar filaments by cell shearing and differential centrifugation. The flagella were visualized by negative stain electron microscopy revealing thin straight or gently curved filaments approximately 11 nm in diameter and up to 10 μm in length. This archaeal flagellum has a diameter significantly smaller than that of bacterial flagella. The M. hungatei flagellin components were separated by SDS PAGE, excised, tryptic digested and analyzed by LC/MS to identify the major flagellin proteins. Unlike the flagella of other described archaeal species, M. hungatei contains only one major flagellin protein, Mhun_3140, one of the three FlaB paralogs present in the genome. On SDS gel, this protein exhibited a molecular weight significantly higher than that predicted from its amino acid sequence, suggesting post-translational modifications. A glycostain and subsequent glycan analysis confirmed the presence of a glycan modification. We have determined a three-dimensional reconstruction of the archaeal flagellar filament at 7.5A resolution by cryo electron microscopy (cryoEM). The structure reveals a core α helix in each subunit that contributes to the formation of the filament that is similar to that observed the bacterial type IV pili. Despite this similarity, the archaeal flagella structure exhibits many non-helical structural elements that bear little resemblance to the bacterial type IV pili.