Giant flagellins form thick flagellar filaments in two species of marine γ-proteobacteria

Abstract

Flagella, the primary means of motility in bacteria, are helical filaments that function as microscopic propellers composed of thousands of copies of the protein flagellin. Here, we show that many bacteria encode "giant" flagellins, greater than a thousand amino acids in length, and that two species that encode giant flagellins, the marine γ-proteobacteria Bermanella marisrubri and Oleibacter marinus, produce monopolar flagellar filaments considerably thicker than filaments composed of shorter flagellin monomers. We confirm that the flagellum from B. marisrubri is built from its giant flagellin. Phylogenetic analysis reveals that the mechanism of evolution of giant flagellins has followed a stepwise process involving an internal domain duplication followed by insertion of an additional novel insert. This work illustrates how "the" bacterial flagellum should not be seen as a single, idealised structure, but as a continuum of evolved machines adapted to a range of niches.

Fig 1. A phylogeny of giant flagellins.

Phylogeny generated from conserved D0/D1 domain alignments; circles at ends of branches depict the size of the insertion, labels denote species with length of the full flagellin gene product in parentheses. Colours of circles represent discrete homology groups. The red star denotes the region in which B. marisrubri and O. marinus are found. Scale bar denotes average number of amino acid substitutions per site. Species are representative of diverse phyla, including α-proteobacteria (Methylobacterium sp.), γ-proteobacteria (Acinetobacter sp) and δ-/ ε-proteobacteria (Desulfotalea psychrophila), Aquificae (Thermosulfidibacter takaii), Planctomycetes (Blastopirellula marina), Synergistetes (Aminomonas paucivorans) and the Firmicutes (Eubacterium plexicaudatum).

Fig 2. The Oceanospiralles giant flagellin forms a thick filament and evolved via an internal duplication.

(A) TEM images of B. marisrubri and O. marinus. The images are typical of the cell morphology and flagellation pattern that we observed for each strain. (B) Negatively stained TEM images of representative flagellar filaments from B. marisrubri and O. marinus. A filament (24 nm diameter) from S. Typhimurium is also shown for comparison. All images are to the same scale. (C) Schematic depicting the phylogeny and occurrence of DE regions in related organisms. The phylogeny was determined using only the N- and C-terminal sequences that make up the D0 and D1 domains. Domain Extension (DE) regions are depicted in green; the C-terminal duplication is depicted in teal green. Arrows and accompanying grey boxes indicate inferred points at which the DE region was duplicated (“i”) and at which an additional insert was added between DE₁ and DE₂ (“ii”). Scale bar denotes average number of amino acid substitutions per site.

Fig 3. A model for evolution of the Oceanospiralles giant flagellin.

Two possible scenarios for protein folding are conceivable: (A) the DE region duplicates and the two discrete DE sequences fold into discrete domains. Subsequent generation of the DX insert would make a more bulky flagellin. (B) Alternatively, the DE region duplication leads to two split domains, wherein the two physical DE regions are comprised of disparate parts of the two contiguous DE sequences. Subsequent insertion of the DX insert would make a longer protrusion at the surface.