Systems biology knowledge is crucial for our understanding of the cellular and molecular basis of medicine and synthetic biology applications. It also provides a framework for metabolomic studies of various biological systems. Systems biology knowledge will advance through investigations that confirm computational models of cellular physiology or discover new pathways not previously described. Using E. coli K12 as a model system for cellular physiology studies is optimal due to its biological simplicity and genetic tractability. One crucial element missing from bacterial cell physiology studies is precise mechanisms defining cross-talk between two different stress responses. Stress responses are rapid physiological adaptations to environmental changes. Bacterial small regulatory RNAs are noncoding RNA molecules with a post- transcriptional regulatory function. Small RNAs frequently act as genetic switches, by affecting specific metabolic pathways in major ways, via post-transcriptional regulation of a central regulator or enzyme involved in a pathway. For this reason, the study of small RNAs is uniquely suited for studies aimed at defining physiological circuits and cross-talk. The small regulatory RNA RybB is regulated by the Extracytoplasmic function (ECF) sigma factor, RpoE, in E. coli. RpoE is a central regulator of the envelope stress response in E. coli and is analogous to the unfolded protein response in eukaryotic endoplasmic reticulum. NsrR responds to nitric oxide (NO) exposure by inducing the expression of HmpA, a NO-detoxification enzyme. Hence, NsrR is a central mediator of the nitrosative stress response in E. coli. We have preliminary evidence to suggest that a nitric oxide sensing transcriptional regulator, NsrR, has a regulatory effect on RybB expression as well. The implications for this observation is that there may be physiological cross-talk between the nitrosative stress and envelope stress responses in E. coli; and, it can be studied by characterizing the role NsrR plays on RybB. Our main hypothesis is that NsrR is a direct regulator of RybB expression. We have two models we propose as a mechanism for our hypothesis that NsrR directly regulates RybB. Our first model is that NsrR exerts its regulatory effect on RybB via an interaction with DNA, specifically the promoter of the rybB gene. Our second model is that NsrR exerts its regulatory effect on RybB via a direct interaction with the RpoE protein, acting as an anti-sigma factor and preventing it from regulating is transcriptional targets (including rybB). To test our hypothesis, we will first determine the regulatory level at which NsrR acts on RybB expression (Specific Aim #1). Then, we will identify biochemical interactions necessary for the NsrR effect on RybB expression (Specific Aim #2). Finally, we will determine the role that exogenous NO exposure plays on the expression of RybB (Specific Aim #3). Taken together, these experiments will characterize NsrR's regulatory effect on RybB and contribute to knowledge of physiological cross-talk in bacterial cells. The models resulting from these studies will contribute to refined physiological models for systems biology applications.
Public Health Relevance: Bacterial infections remain a global public health threat and their remains a dire need to increase our knowledge of their adaptive capabilities. The envelope stress response and nitrosative stress responses are both important pathways, critical in defining bacterial adaptation to the innate immune response. Understanding the mechanisms that bacteria use to adjust to nitric oxide exposure will hasten our understanding of bacterial cell biology and increase our ability to create novel therapeutics.
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