Bacillus subtilis is able to respire with either oxygen (aerobic respiration) or nitrate (anaerobic respiration). Both forms of respiration require the ResD-ResE two-component signal transduction system. ResD, phosphorylated by ResE sensor kinase, activates transcription of genes required for aerobic and anaerobic respiration. This project will uncover the regulatory mechanisms by which B. subtilis commits to undergo anaerobic respiration. Two regulatory proteins NsrR and unphosphorylated ResD play critical roles in the decision-making process. NsrR is a Fe-S transcription regulator that represses anaerobic respiration genes but the repressor activity is eliminated in response to nitric oxide (NO) produced during nitrate respiration. This research determines how NO modifies the Fe-S cluster in NsrR and how this modification alters NsrR activity. Unphosphorylated ResD activates anaerobic respiration genes in response to oxygen limitation. Two hypotheses will be tested: One, that ResD itself activates transcription or, two, that an accessory factor interacting with ResD and/or the promoter DNA is responsible for transcriptional activation. To investigate these possibilities, mutations that affect unphosphorylated ResD-dependent activation will be isolated and characterized. The responses of microorganisms to oxygen limitation and to NO greatly impact the activities involved in the earth's nitrogen cycles and in pathogenesis. Both NsrR and two-component signal transduction proteins are widely present in bacteria. The research takes advantage of a powerful bacterial genetic system and leading-edge metallobiochemistry to gain new insights into these ubiquitous regulators and their associated mechanisms.
Broader impact of this project is multidisciplinary research training in bioinorganic chemistry and molecular genetics for a variety of groups. These diverse groups include not only Ph.D. students but also non-thesis M.S. students who, after completing one-year training in scientific research and education, continue their careers in graduate school (Ph.D.), medical school, or as research technicians in both academic and industrial laboratories. The project will also train undergraduates supported by summer internship opportunities offered by the department and REU supplement funds, high school students supported through Saturday Academy, and high school teachers supported by Partners in Science Program of M.J. Murdock Charitable Trust, in which the P.I. has participated.
All living organisms from unicellular bacteria to humans constantly encounter a wide variety of environmental changes, to which they must quickly adapt. Often, this involves sensing the presence of toxic agents, either present externally or generated internally as a result of metabolic imbalances induced by environmental insult. How organisms deal with toxic conditions is a critical question that must be answered in order to understand the biological impact of problems ranging from infectious disease to global climate change. Bacteria grow and thrive within a wide variety environmental niches and often under harsh conditions. Hence, they serve as excellent model systems for the study of this fundamental question about life. The research has uncovered how a soil bacterium Bacillus subtilis responds to oxygen limitation and nitric oxide (NO, a toxic radical) and alters gene expression in order to shift its metabolism to cope with the drastic environmental change that affects cellular energy generation. The responses of microorganisms to oxygen limitation and to NO greatly impact the activities involved in the earth’s nitrogen cycles and in bacterial pathogenesis. In addition, NO is a key signaling molecule in human physiological processes. As NO is easily diffusible, highly reactive, and short-lived, it is suitable as a signaling molecule. Using molecular genetic, biochemical, and biophysical approaches, the study uncovered how the NsrR transcriptional regulator senses NO and how interaction of NO with NsrR alters gene expression controlled by NsrR. The grant also provided students at all educational levels including Ph.D./M.S. students, summer-time undergraduate, and high school student interns with unique opportunities for interdisciplinary research involving leading-edge technology. Furthermore, a high school teacher has been involved in the experimental work and learned up-to-date scientific knowledge and techniques, which was applied to classroom curriculum.
