<p>Bacteria adapt to changes in their environment by regulating gene transcription. TetR family transcriptional regulators (TFRs) constitute one of the largest groups of bacterial transcription factors and thus, characterization of TFRs is anticipated to be crucial for a better understanding of prokaryotic physiology. Of significant importance, the majority of TFRs are predicted to respond to small-molecule signals and an emerging paradigm suggests that identifying ligands of TFRs can provide direct insight into the biochemical functions of the genes they regulate. Regulatory target genes and small-molecule ligands are unknown for all but a few TFRs and therefore, generally applicable tools for identifying these basic elements of TFRs are highly desirable. We first investigated the use of genome context as a predictive tool for identifying regulatory targets of TFRs. We find that the majority of TFRs are divergently oriented from a neighboring gene, and those with a“200 bp rule” should allow us to predict at least one regulatory target for more than half of all TFRs in the public databases. Second, we developed a biosensor mechanism amenable to high-throughput screening for identifying ligands of TFRs of unknown function. Significantly, one of our biosensors has played an integral role in characterizing the ligands of a previously uncharacterized TFR. Thus, the combined use of the tools we have developed will provide considerable benefit in understanding bacterial small-molecules responses mediated by TFRs.</p> / Doctor of Philosophy (PhD)