Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei

Area of Science:

• Microbiology
• Archaea
• Cellular Motility

Background:

• Methanospirillum hungatei, an archaeon, exhibits phenotypic plasticity in response to environmental phosphate concentrations.
• Cellular morphology and motility in M. hungatei are significantly altered by varying phosphate availability in growth media.

Purpose of the Study:

• To investigate the impact of phosphate concentration on the flagellar apparatus and motility of Methanospirillum hungatei.
• To characterize the protein composition and post-translational modifications of M. hungatei flagella.

Main Methods:

• Cultivation of M. hungatei strains GP1 and JF1 in media with varying phosphate concentrations (3, 30, and 45 mM).
• Electron microscopy for visualizing cell morphology and flagellar structures.
• Flagellar filament isolation, protein analysis using SDS-PAGE, and Western blotting with specific antisera.
• Chemical deglycosylation to assess glycosylation of flagellins.

Main Results:

• Low phosphate (3-30 mM) induced motile cells with polar flagellar tufts, while high phosphate (45 mM) resulted in nonmotile cells lacking flagella.
• Flagellar filaments, approximately 10 nm in diameter, were sensitive to detergents and composed of two major flagellins (Mr 24,000 and 25,000).
• Strain JF1 produced a third flagellin (Mr 35,000) under specific trace element conditions, and all flagellins were identified as glycosylated.

Conclusions:

• Phosphate availability is a critical regulator of flagellar expression and motility in Methanospirillum hungatei.
• The flagella of M. hungatei are composed of at least two, and potentially three, distinct glycosylated flagellins, with limited serological cross-reactivity between strains at the Mr 24,000 flagellin level.