Probing the Metabolism and Cellular Roles of 2',3'-Cyclic Nucleotide Monophosphate

Abstract Novel intracellular small molecules, 2’,3’-cyclic nucleotide monophosphates (2’,3’-cNMPs), have recently been discovered within both prokaryotes and eukaryotes. Within plants and mammals, wounding has been found to cause increased levels of 2’,3’-cNMPs. Preliminary studies in bacteria suggest that 2’,3’-cNMPs also are produced in response to cellular stress and that 2’,3’-cNMP levels affect bacterial phenotypes, such as biofilm formation and motility, and expression of numerous genes. The long-term goal of this research is to understand the roles of 2’,3’-cNMPs in controlling prokaryotic signaling pathways and to utilize this knowledge to design small molecules to modulate cellular phenotypes. This goal will be addressed by investigating the cellular components involved in 2’,3’-cNMP metabolism, identifying 2’,3’-cNMP sensors, and determining the effects of 2’,3’-cNMPs on downstream pathways in a wide range of bacteria. The proposed work outlines an innovative research plan to probe a novel cellular stress-sensing mechanism and will provide molecular level details about the nucleic acids and proteins involved in 2’,3’-cNMP metabolism, as well as the downstream cellular phenotypes. The proposed studies also will expand knowledge of cellular mRNA decay pathways within bacteria because 2’,3’-cNMPs are products of mRNA degradation. This work is anticipated to yield the following expected outcomes. First, it will identify the proteins responsible for 2’,3’-cNMP production and degradation in vivo, as well as 2’,3’-cNMP binding proteins that may control downstream phenotypes. These studies also will highlight the distribution of 2’,3’-cNMPs within the bacterial kingdom and extend our understanding of mRNA decay within prokaryotes. Second, the proposed work will identify the effects of altering 2’,3’-cNMP levels in an array of bacteria, including changes in gene expression, phenotypes, and key metabolic pathways, including nucleotide/nucleoside metabolism. Third, elucidating conditions that alter 2’,3’-cNMP levels and phenotypes controlled by 2’,3’-cNMPs will highlight their role in bacterial responses to cellular stress, particularly with regards to cellular proliferation and biofilm formation, and illuminate additional effects of stress on mRNA decay. The proposed work will have an important positive impact by dissecting the cellular roles of 2’,3’-cNMPs within bacteria, which will highlight novel pathways within prokaryotes and, in the future, potentially can be engineered to control bacterial proliferation.