Signal-Transduction Mechanisms of O2-Sensing c-di-GMP Phosphodiesterases

University Of Texas Southwestern Medical Center, Dallas TX

Investigators

Abstract

The broad objectives of this research are to determine how biological systems adapt to changes in oxygen tension and how biosensors mechanistically detect oxygen. The project focuses on three proteins that govern bacterial adaptations to oxygen fluctuations: Acetobacter xylinum PDEA1, Escherichia coli Dos, and Escherichia coli YddV. All three accomplish their functions by adjusting the cellular levels of cyclic bis 3'-5' diguanylic acid (c-di-GMP), a small dinucleotide that governs the transitions of bacteria between motile and sessile states. PDEA1 controls the production of a cellulose biofilm by A. xylinum based on oxygen availability; this protein closely resembles E. coli Dos in that it features a heme-binding oxygen-sensor module of the Per-ARNT-Sim (PAS) family together with a c-di-GMP degrading phosphodiesterase of the GGDEF/EAL class. In E. coli, a c-di-GMP manufacturing protein YddV is produced jointly with Dos from a single operon and is expected to partner with Dos in governing motility changes in response to oxygen fluctuations. The project addresses the following questions: What are the physiological roles of the yddV-dos operon and how are the opposing functions of YddV and Dos manifested in E. coli? What strategies are available to oxygen sensors for establishing their sensing threshold? Once this threshold is reached, how do these heme-based sensors "switch" their coupled enzymatic activity? The research will combine microbiological, molecular genetic, biochemical, and biophysical approaches, such as preparation and examination of bacterial gene knockouts, site-directed mutagenesis, measurement of enzymatic parameters, and determination of ligand-binding parameters. Data from this project will extend understanding of the importance of Dos and YddV to E. coli, the factors that influence binding of gaseous signals to biosensors, and the biochemical mechanisms used for regulating their activities. Undergraduates from the University of Texas at Dallas will be integrated in the project together with doctoral and postdoctoral researchers from University of Texas Southwestern Medical Center at Dallas, so that the work is conducted in an environment that is diverse in its skills, levels of training, and human make up. Intellectual merits of the research are that it will expand knowledge of signal-transduction mechanisms, the roles of nucleotide second messengers, and the regulation of bacterial biofilms. A broader impact will be the training of a high proportion of normally underrepresented minorities at the graduate, undergraduate, and post-doctoral level.