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The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid β-alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah-BarR. Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah-BarR is a dual-function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah-BarR protein. β-alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C-terminal tail in Ah-BarR. By performing site-directed mutagenesis, this tail is shown to be involved in ligand-binding response., (© 2023 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion. Here, an approach is described for the selection and cloning of TF-promoter pairs, the development of a reporter system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is illustrated together with prospective adaptations to modify a reporter system's output signal. The given approach can be used for the investigation of native, heterologous, or even artificially created TFs in Escherichia coli, and can be extended toward use in other microorganisms., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)