Structural and functional insights into dual-signal recognition by a LysR-type transcriptional regulator in Agrobacterium tumefaciens

The soil-borne plant pathogen Agrobacterium tumefaciens is the causal agent of the crown gall disease in plants. Its transition between host and soil environments depends on tightly regulated response systems. A crucial factor in environmental adaptation of A. tumefaciens is the LysR-type transcriptional regulator (LTTR) LsrB. Deletion of lsrB leads to misregulation of over 1,200 genes, which results in pleiotropic phenotypes such as reduced virulence and motility and increased sensitivity to antibiotics and reactive oxygen species (ROS) (1–3). Recent results from our group indicate that LsrB senses two distinct signals: ROS and the plant-derived signal molecule acetosyringone. In the present study we focus on refining the structural and functional mechanisms underlying LsrB-dependent regulation.

Size exclusion chromatography and in vivo crosslinking revealed that LsrB assembles as a homotetramer, consistent with other known LTTRs(4). AlphaFold3 modelling of the LsrB structure suggests that this regulator harbors I) redox-responsive cysteines and II) a conserved ligand-binding site, putatively involved in the recognition of the two distinct signals. Therefore, we performed site-directed mutagenesis of amino acids likely involved in signal recognition and assessed their contribution to LsrB function in vivo by phenotypic assays. Further, in vitro thiol trapping coupled to mass spectrometry support our model of cysteine-based redox sensing. Currently we are carrying out cryo-EM to resolve the LsrB structure and elucidate the precise signal recognition mechanism. Taken together, we present evidence for LsrB"s dual-signal detection mechanism and contribute to the knowledge of signal-driven regulation by LTTRs.

Tang, G. et al. The LsrB Protein Is Required for Agrobacterium tumefaciens Interaction with Host Plants. Mol. Plant-Microbe Interactions® 31, 951–961 (2018).Schmidt, J. J. et al. The LysR-type transcription factor LsrB regulates beta-lactam resistance in Agrobacterium tumefaciens. Mol. Microbiol. 121, 26–39 (2024).Eisfeld, J. et al. A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens. Mol. Microbiol. 116, 126–139 (2021).Baugh, A. C., Momany, C. & Neidle, E. L. Versatility and Complexity: Common and Uncommon Facets of LysR-Type Transcriptional Regulators. Annu. Rev. Microbiol. 77, 317–339 (2023).