Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen.

The R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for transcription factors. Comparative bioinformatics approaches identified and experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in -based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria. spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the response regulator RR_1586 by first screening for protein-DNA interactions in and then predicting regulatory outputs in .