Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe acute and chronic lung infections in patients with a compromised immune system. It is a mono-flagellated bacterium and the flagella motility is vital for virulence, pathogenesis and biofilm formation. Flagella are also important for spread of the bacterium from the site of infection. Bacteria that lose their flagellum are far less virulent, showing how critical this structure is for infection and disease progression.
The flagellar filament is primarily composed of a protein called flagellin [1]. Production of flagellin is controlled by a specialized regulatory protein called FliA (also known as σ28) which works together with RNA polymerase (RNAP) to switch on genes required for bacterial movement. FliA controls many motility-related genes, acting like a master ‘on-switch’. When the fliA gene is disrupted, the bacteria become completely non-motile.
FliA itself is tightly regulated by its cognate anti-sigma factor, FlgM, which binds to FliA and prevents its association with the RNAP core enzyme, acting as a natural “off-switch”. Changes in the balance between these two proteins strongly affect bacterial movement: Deletion of the FlgM gene causes excessive flagellin production but results in a weakly motile phenotype, while overexpression of FlgM produces a non-motile phenotype. The aim of this work was to understand, at the molecular level, how these proteins regulate bacterial motility and virulence. Using the ESRF beamlines, ID30a and CM01, we solved the high-resolution crystal structure of the FliA-FlgM complex and further determined the Cryo-EM structure of the FliA-RNAP-DNA assembly, revealing several unique structural features during transcription activation [2].
The structure of the FliA-FlgM complex reveals that FlgM tightly wraps around FliA, extensively contacting all three domains and locking it in an inactive form that cannot bind RNA polymerase or promoter DNA (Figure 1A). By masking the interaction surfaces required for transcription, FlgM effectively switches off flagellin synthesis and bacterial motility.
The Cryo-EM structure reveals major conformational changes in FliA upon release from FlgM, enabling its association with the catalytic core of RNA polymerase to turn on flagellar genes (Figure 1B). It also uncovers how FliA specifically recognizes the -35 promoter region, allowing correct positioning of the -10 element where DNA strand opening begins (Figure 1C). At this stage, a conserved arginine helps flip DNA bases at position -11, helping to separate the DNA strands and begin forming the transcription bubble, highlighting a unique mechanism of DNA melting by FliA (Figure 1D). Another key observation is that the linker between domains 3 and 4, known as the sigma finger, is only partially engaged in the RNAP active site, and the transcription bubble remains partially formed, suggesting the structure captures an intermediate state en route to full transcription activation.
Mutations of the key residues identified in both structures impair transcription in vitro and reduce motility in vivo, validating the structural findings and their functional significance. These findings provide a detailed mechanistic understanding of anti-sigma factor mediated inhibition, in which FlgM acts as a molecular checkpoint to tightly regulate motility gene expression. Furthermore, the structural insights into σ28-dependent promoter recognition and transcription initiation significantly advance our understanding of transcription regulation by flagellar sigma factors. More broadly, these insights can be used to design novel synthetic genetic circuits or development of inhibitors that can block flagellation and consequently reduce virulence in Pseudomonas aeruginosa.
D. Jain (RCB, India), E. Kandiah (ESRF)
[1] Sheenu and Jain D (2025) Biochemistry, 64, 770-781
[2] Sheenu, Kumar V, Sahoo PK, Kandiah E and Jain D. (2026) Nucleic Acids Res, 54, gkaf1414
Figure 1: (A) X-ray crystal structure of FliA (blue) -FlgM (pink) complex, showing FliA in compact inhibited state. (B) Single particle Cryo-EM structure of the FliA-RNA polymerase-DNA complex revealing three unique structural features including (C) base-specific interactions at the -35 promoter region (D) flipping of template and non-template -11 bases (black) and incomplete insertion of spacer (magenta) of FliA in the active site. The DNA is shown in ribbon representation, with the template strand in orange and the non-template strand in yellow, while the core RNAP is displayed in surface representation in green.