Distinct flagellins differentially fine tune biofilm initiation via flagellar stator-associated proteins

<i>Helicobacter pylori</i> relies on flagellar motility to colonize the gastric niche. While <i>H. pylori</i> flagella serve multiple functions, including swimming motility, it has recently been appreciated that flagellar basal body properties also contribute to biofilm initiation. The <i>H. pylori</i> flagellar filament contains two spatially segregated flagellins, FlaA and FlaB, but their roles in the biofilm initiation process remained undefined. Here, we confirmed that both FlaA and FlaB are required for optimal motility, but found that they exert opposite effects on biofilm initiation: cells without FlaA display low biofilm initiation, while cells without FlaB display elevated initiation. To understand the molecular basis for this divergence, cryo-electron microscopy (cryo-EM) analysis of native flagellar filaments revealed that FlaA and FlaB have distinct supercoiled waveforms. We confirmed these intrinsic waveform curvatures by analyzing filaments from Δ <i>flaA</i> and Δ <i>flaB</i> mutants, providing a physical basis for their functional specialization. Genetic epistasis experiments further demonstrated that the enhanced early biofilm formation of the Δ <i>flaB</i> mutant depends on PilO, a stator-associated component of the flagellar motor recently shown to drive a reciprocal biofilm and motility response. Our findings establish that <i>H. pylori</i> has developed functionally specialized flagellins that work with motor-dependent signaling to dynamically balance surface colonization and motility.