Mechanisms of pterin-dependent regulation in proteobacterial systems

PROJECT SUMMARY The transition of bacteria from a free-living state to a sessile, surface-attached growth mode often leads to formation of multicellular biofilms, profoundly altering cellular physiology and increasing antimicrobial tolerance. Attachment and biofilm formation is regulated in many systems by the pivotal cytoplasmic second messenger cyclic diguanylate monophosphate (c-di-GMP), with high levels commonly promoting adherence and low levels favoring planktonic growth. Recent work has identified a remarkable regulatory circuit that controls intracellular c-di-GMP levels through the activity of excreted metabolites called pterins. Diguanylate cyclases (DGCs) synthesize c-di-GMP from GTP, and phosphodiesterases (PDEs) degrade the signal, with certain enzymes that have both activities, the balance of which is often under environmental control. In Agrobacterium tumefaciens, a plant pathogenic Proteobacteria, the DcpA protein is a dual function DGC-PDE, that requires excreted pterins to maintain a PDE-dominant activity in planktonic culture. Most forms of life synthesize pterins from a branch of the folate biosynthesis pathway and in bacteria their excretion is recognized but poorly understood. In A. tumefaciens pterins are released into the periplasm, where they interact with PruR, a pterin binding protein. Pterin-associated PruR interacts with the DcpA periplasmic domain, and biases the protein to a PDE-dominant state. Specific reduced pterins generated by pteridine reductases in the cytoplasm, are preferred ligands for PruR, and mutants that do not produce these reduced pterins switch DcpA from PDE-dominant, to a strong DGC activity, stimulating attachment and biofilm formation. PruR-DcpA regulatory circuits are broadly, but non- uniformly conserved among the Proteobacteria and comparative structural analysis predicts that most of the PruR-type proteins will bind pterin-type molecules. The proposed study is designed to test and expand the current PruR-DcpA model in A. tumefaciens, as a prototype for related bacterial systems, using a combination of molecular genetics, biochemistry, synthetic organic chemistry, genomics and bioinformatics. The research plan will perform in depth interrogation of pterin-PruR binding and determine how this in turn regulates DcpA activity. The production and excretion of pterins as a branch of the folate pathway, and to what extent this impacts PruR-DcpA regulation, will be studied using antibiotics, targeted genetic manipulation, and an unbiased mutant screen. The phylogenetic distribution of PruR-DcpA-type systems will be extensively investigated, elucidating additional genetic relationships, patterns of conservation, and paths of evolution. Expanding beyond the A. tumefaciens system, a genetically tractable opportunistic human pathogen, Aeromonas veronii with four PruR-DcpA modules, will be dissected using heterologous expression, genetics, and biochemical approaches to disentangle their regulatory roles. This work will provide numerous insights into this newly discovered, widely relevant mechanism controlling biofilm formation and likely other yet to be identified processes, with the potential to lead to new therapeutic targets.