Characterization of LeuO and additional LysR-type transcription regulators in Escherichia coli

• Applicant: Professorin Dr. Karin Schnetz
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2017 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 392159619

LysR-type transcription regulators (LTTRs) constitute the most abundant family of transcription regulators in Bacteria and are implicated in regulation of a broad spectrum of processes related to stress responses, metabolism, and pathogenicity. LTTRs consist of an N-terminal winged helix-turn-helix (wHTH) DNA-binding domain that is connected with a helical linker to a C-terminal effector-binding domain (EBD). LTTRs are tetramers which contain two dimeric wHTH-linker domains and specifically contact DNA at two sites. Effector-binding induces a structural change that triggers a shift of one of these contacts along the DNA (sliding dimer mechanism) and thus a concomitant change in the binding pattern which is relevant for the effector-specific regulation of target genes, when the LTTR is present at a physiological low level. However, the sliding dimer mechanism of regulation and the dependence of the specificity on the cellular level has not been considered in many studies on LTTRs of pleiotropic function. With this proposal the pleiotropic LTTR LeuO in Escherichia coli, as well as additional LTTRs, shall be functionally characterized in respect to activity, mechanism of transcription regulation, and pleiotropic function. LeuO regulates more than 100 target loci and is considered an antagonist of the global nucleoid-associated repressor proteins H-NS and StpA. However, up to date it is unknown how LeuO activity is modulated, how LeuO specifically binds DNA, and how LeuO regulates transcription and acts as H-NS and StpA antagonist. In preliminary work we have solved the crystal structure of the LeuO-EBD and isolated hyperactive mutants that presumably mimic an effector-induced state. With this proposal we plan to characterize the DNA-binding specificity by comparing wild-type LeuO, hyperactive LeuO mutants, and the dimeric wHTH-linker domain, which is sufficient for activation of target loci. In addition, ChIP-seq and RNA-seq with a hyperactive LeuO variant will be used to re-evaluate the spectrum of target loci. Second, we wish to establish a structure-function relationship of LeuO by solving the crystal structure of full length LeuO and by a mutational analysis which will include genetic and biochemical assays. Replacement of specific amino acid residues by crosslinkable unnatural amino acids shall identify interactions relevant for transcriptional regulation (as with RNA-polymerase and/or H-NS and StpA). Third, the mechanism of regulation as H-NS and StpA antagonist shall be addresses in vivo. To this end, LeuO will be tagged with repeats of a split-superfolder-gfp epitope (for fluorescence signal enhancement) and then used for co-localization studies with H-NS. With these two approaches (crosslinking and co-localization) the question whether LeuO co-binds with H-NS or displaces it from the DNA may be clarified. This working pipeline, established for LeuO, will be applied for characterization of the functional role of other LTTRs in E. coli.

• DFG Programme: Research Grants