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.
Highlights
Protein methylation plays a key role in physiological function and signaling pathway modulation
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
Summary
Protein methylation plays a key role in physiological function and signaling pathway modulation. The first protein methylation was reported for lysine residues in the flagellin of S. enterica in 1959 by Ambler and Rees [1]. Flagellin is the protomer (subunit) of the bacterial flagellar filament, which is a tubular supercoiled polymer assembled by ~20,000 flagellin subunits. The flagella protrude from the bacterial cell body and propel the bacterium for targeted movements. Flagella play important roles in host cell surface adhesion, colonization, biofilm formation and host inflammatory activation during infection [5,6,7,8]. The structure of the flagellin monomer is organized into four connected domains: the highly conserved D0 and D1 domains at both the N- and C-terminus forming the inner core of the filament structure and the variable D2 and D3 intervening domains forming the outer surface of the filament [9,10,11] (S1 Fig)
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