Nitric oxide regulation of c-di-GMP metabolism and biofilm formation in Shewanella woodyi
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Biofilms, multi-cellular sessile communities of bacteria, are known to account for bacterial persistence and antibiotic resistance. Nitric oxide (NO) has been shown to induce biofilm dispersal at sub-lethal concentrations in many species. For example, in the cystic-fibrosis associated bacterium, Pseudomonas aeruginosa, NO is reported to regulate biofilm dispersal through cyclic di-GMP signaling. In this thesis work, I demonstrate that Swoo_2750 from Shewanella woodyi encodes a Heme-Nitric oxide/OXygen binding domain (H-NOX), a protein that binds NO with approximately picomolar sensitivity. I demonstrate further that SwH-NOX is co-cistronic and directly interacts with Swoo_2751, a bi-functional diguanylate cyclase (DGC), which exhibits both c-di-GMP synthesis and hydrolysis activities. Through steady-state kinetic analyses, I conclude that NO bound H-NOX interacts with DGC and induces a 15-fold increase in c-di-GMP hydrolysis as well as a 90% decrease in c-di-GMP synthesis compared to the effect on DGC found with unligated H-NOX. I have correlated these biochemical data with measurements of in vivo c-di-GMP concentrations and assessments of biofilm formation, both in wildtype and a mutant (Δhnox) strain of S. woodyi. These studies lead to the conclusion that this H-NOX signaling pathway provides a molecular-level explanation for the rapid dispersal of biofilms that has been observed in the presence of NO. Finally, I have explored the source of NO used in H-NOX signaling, especially the role of anaerobic respiration in regulation of biofilm formation in S. woodyi. In summary, I present here my effort towards understanding nitric oxide signaling in biofilm regulation.