Lysinoalanine crosslinking in the extracellular flagellar hook of Synergistota

ABSTRACT Bacterial motility is driven by the flagellar motor, a conserved macromolecular complex that enables cells to navigate chemical gradients. In spirochetes, motility depends on periplasmic flagella whose function is supported by a unique post-translational modification in the hook protein FlgE: a covalent lysinoalanine (Lal) crosslink between adjacent subunits. This modification has been shown to be conserved across the phylum and is required for motility. In spirochetes, Lal is thought to provide additional hook stability needed to meet the demands of flagellar rotation in the confined space of the periplasm and associated load of the wrapped cell body. Guided by a bioinformatic screen based on conserved catalytic residues required for Lal formation, we investigated the possibility that bacteria with extracellular flagella also produce Lal. Using a combination of biochemical crosslinking assays and high-resolution mass spectrometry, we demonstrate that FlgE from Thermanaerovibrio acidaminovorans, a motile member of the Synergistota phylum, forms Lal crosslinks analogous to those of spirochetes in vitro and in wild-type cell culture. These results establish the first example of Lal crosslinking in extracellular flagellar hooks and the first instance outside of the spirochete phylum. Thus, Lal crosslinking is more broadly conserved than previously appreciated and may represent a general mechanism for enhancing hook stability and function for extracellular as well as periplasmic flagella. IMPORTANCE Many bacteria employ complex rotary engines known as flagella to propel them toward nutrients or away from harmful environments. In spirochetes, motility depends on lysinoalanine (Lal) crosslinks within the flagellar hook protein FlgE to strengthen the universal joint of the rotating filament, which resides in the periplasm. This study shows that the same type of modification also exists in a bacterium distantly related to spirochetes, Thermanaerovibrio acidaminovorans , that contains more typical extracellular flagella. Using biochemical experiments and mass spectrometry, we confirm that its flagellar hook protein self-catalyzes Lal crosslinks, marking the first time this modification has been observed outside spirochetes and in extracellular flagella. These findings suggest that Lal crosslinking is more widely conserved than previously thought and may represent a general strategy bacteria use to reinforce their flagella. Because motility is closely linked to survival and the ability of bacteria to cause disease, this discovery improves our understanding of bacterial motility mechanisms and how it is maintained across diverse species.