This project aims at understanding of the transcriptional regulation in Bacillus subtilis that is responsive to oxygen limitation. The ResD-ResE two-component signal transduction system and the NO-sensitive NsrR repressor play key roles in controlling transcription of genes involved in anaerobiosis. B. subtilis NsrR contains a [4Fe-4S] cluster and represses transcription of ResD-controlled nasDEF (nitrite reductase genes) and hmp (flavohemoglobin gene). Interaction of NO with the [4Fe-4S] cluster alleviates binding of NsrR to the nasD and hmp promoters, leading to derepression (class I regulation). NsrR also plays a role in more global transcription by interacting with A+T-rich DNA in an NO-insensitive manner at least in vitro (class II regulation). Class II genes are upregulated by an nsrR mutation when pyruvate is present, and this upregulation is ResD-dependent. This research will investigate a critical link between ResD and NsrR, two major transcriptional regulators that play pivotal roles in anaerobiosis of B. subtilis. As binding of NsrR to class II genes shows relaxed sequence specificity and a target bias that likely involves DNA topology, the in vivo interaction of NsrR with the promoter regions of class II genes will be determined. Sensitivity of DNA interaction to NO will be compared between class I and class II promoters in parallel with evaluation of NO effects on transcription of corresponding genes. Transcription of class II NsrR-controlled genes is often regulated by other transcription factors, such as ResD and AbrB, that also target A+T rich DNA. This research will characterize how multiple transcription factors affect NsrR binding at specific target genes. Studies of a novel regulatory mechanism of NsrR and accompanying NsrR-dependent global regulation will advance our understanding of transcriptional control in response to oxygen limitation and how multiple transcriptional regulators control expression of selected genes in response to a variety of environmental signals.
Broader Impacts: A postdoctoral fellow, thesis/non-thesis MS students, undergraduate interns, and a high school teacher will participate in the research. The project will train them in the scientific method, to acquire knowledge through experimentation, and to gain further experience in a research environment while training in modern techniques involving genetics, molecular biology, biochemistry, and physiology. The postdoctoral fellow will incorporate his/her training as a researcher and as an educator by directly mentoring the MS students and undergraduate interns. The high school teacher will use the research experience to revitalize his/her teaching in the classroom. Multi-tiered mentoring will be implemented and the P.I. will work to establish productive interactions among participants. A summer internship for undergraduates will be jointly organized with CMOP through funding from NSF. The summer internship program offers many activities including a weekly meeting, presentation of research, seminars, and water sampling tours in the Columbia Estuary.
