Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

Abstract

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family. Author summary The bacterial flagella are tail-like appendages that play important roles in motility and host cell infection. In Salmonella, the surface of the flagellar filaments is heavily modified by methylation on their lysine residues, contributing to efficient gut colonization and successful invasion of the host. However, little is known about the properties of the methylase FliB, that catalyzes flagellar methylation. In this study, we isolated and characterized FliB of S. enterica for the first time. We show that FliB forms oligomers that localize in close proximity to the bacterial cell membrane. The FliB methyltransferase coordinates a [4Fe-4S] cluster at its active site by several cysteine residues that were highly conserved in many entero-pathogens. We further found that FliB modifies flagellar subunits using an iron-sulfur cluster dependent mechanism and requires a reducing environment. Our work sets a stage for understanding the roles of oxygen sensitive iron-sulfur cluster in flagellar modification and entero-bacterial pathogenesis.