Bacterial flagella are whip-like structures that drive motility, allowing bacteria to move toward favorable conditions or away from harm through chemotaxis [1]. Their biosynthesis is tightly regulated to conserve energy and respond to environmental signals. A hierarchical regulatory cascade governs flagellar gene expression, with the flhDC operon serving as the master regulator of assembly. Upstream transcriptional regulators and environmental factors further adjust flhDC activity, enabling precise sequential expression of downstream genes for proper flagellar construction [2]. This complex regulation allows bacteria to adapt motility to environmental changes, crucial for survival and pathogenesis, however, it has not yet been fully elucidated.

The LysR-type transcriptional regulator (LTTR) family is one of the largest and most conserved groups of transcriptional regulators in prokaryotes, with orthologues also found in archaea and eukaryotes [3], [4]. LTTRs are known for their conserved DNA-binding domains and initial roles as transcriptional activators of divergently transcribed genes with negative autoregulation. LTTRs are commonly regulated by small-molecule ligands that bind to the C-terminal regulatory domain (RD) and modulate DNA binding and transcriptional output [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. Historically, activating ligands have been referred to as “co-inducers”, whereas the term “ligand” more generally denotes any molecule capable of binding the RD. In most LTTRs, ligands are endogenous metabolites or environmental/host-derived compounds, although in a limited number of cases, metal ions, light sensitivity or redox-dependent modifications have been reported to modulate LTTR activity [4], [15], [16], [17]. Throughout this study, we use the term “ligand” for consistency. LTTRs are involved in a wide array of biological processes, including metabolism, virulence, motility, nitrogen fixation, and stress responses [4].

LysR homologue A (LrhA) is a LysR-type transcriptional regulator with multiple functions [14], [18], [19]. In E. coli, LrhA represses the flhDC operon, the master regulator of flagellar biosynthesis [19], and downregulates type 1 fimbriae genes while influencing rpoS translation [20]. In enterohemorrhagic E. coli, LrhA activates the pchA and pchB genes, which regulate the LEE (locus of enterocyte effacement) virulence genes [21]. Homologues of LrhA in other bacteria play critical roles: RovM in Yersinia promotes biofilm formation and represses the virulence regulator rovA [5], while HexA in Photorhabdus and PecT in Erwinia control exopolysaccharide synthesis and virulence regulation [22]. These findings highlight the pleiotropic regulatory roles of LrhA across bacterial species. However, the ligand for LrhA remains unidentified, and the structural and molecular mechanisms underlying its function and activation are not yet understood.

In this study, we determined the atomic-resolution structure of LrhA through cryo-EM and crystallographic analysis. Structural alignment and comparative studies revealed that the ligand-binding domain of LrhA exhibits significant differences and a lack of conservation compared to previously reported LTTRs. We identified two highly variable regions located at the β5–β6 and β9–β10 linkages as the most distinct structural elements. Mutational studies combined with size exclusion chromatography with multi-angle static light scattering (SEC-MALS) analysis demonstrated that changes of its oligomeric equilibrium caused by mutations in these variable regions impact LrhA oligomeric state. Moreover, fluorescence polarization assays revealed that these mutations significantly enhance the binding affinity of LrhA to downstream promoter DNA. By constructing bacterial mutants, we confirmed that changes in these variable regions shift LrhA toward a more active functional state, leading to enhanced repression of downstream flhDC transcription and regulation of flagellar biosynthesis. Based on these findings, we propose that structural perturbations in the β5–β6 (V1) and β9–β10 (V2) variable regions modulate LrhA activity by shifting its oligomeric equilibrium (tetramer to dimer).