The RNA degradation enzyme RNase E is essential for early flagellar assembly in <i>Escherichia coli</i>

<i>Escherichia coli</i> endoribonuclease E (RNase E), encoded by the essential <i>rne</i> gene and conserved across γ-Proteobacteria, plays a central role in RNA processing and decay. We show here that <i>rne</i>-null strain, <i>rne</i>-null strain complemented with catalytic-null RNase E mutant, and C-terminal-truncated strain (Rned500) all lack flagellar biogenesis and motility under both aerobic and anaerobic conditions, which are restored by wild-type RNase E complementation. The Rned500 displays dysregulated expression of the three-tiered flagellar transcriptional cascade, increased stability of flagellar mRNAs, and reduced flagellar protein levels through sRNA-dependent translational inhibition. However, ectopic expression of flagellar master regulators or flagellar proteins fails to restore flagellar biogenesis and motility. To investigate the underlying defect, we examined the cellular localization of the early flagellar structural protein FliF and found it mislocalized in Rned500, indicating a disruption of early flagellar assembly. This defect is further supported by the impaired secretion of the flagellar anti-sigma factor FlgM in Rned500, a process that requires a functional flagellar basal body. Complementation with wild-type RNase E in Rned500 fully restores expression of the flagellar cascade, proper membrane localization of FliF, flagella formation, and motility. Wild-type RNase E-expressing strains, but not Rned500, activate Toll-like receptor 5 (TLR5)-dependent nuclear factor-kappa B signaling in THP-1 human monocytic cells through flagellin. This response, confirmed by a TLR5 dual-luciferase reporter assay in transfected HEK293T human embryonic kidney cells, highlights RNase E's role in enabling flagellar expression required for cellular immune activation. Collectively, these results identify RNase E as a key flagellar biogenesis regulator, revealing novel posttranscriptional control mechanisms.