FLUORESCENCE-BASED BACTERIAL BIOSENSORS FOR EVALUATING THE INFLUENCE OF WATER STRESS ON BACTERIAL LEAF COLONIZATION
Gwyn A. Beattie* and Catherine Axtell
Department of Microbiology, Iowa State University, Ames, IA

Knowledge of the conditions actually sensed by microorganisms in most terrestrial ecosystems is currently limited by the inability of environmental monitoring tools to function on the scale of a microorganism. We have constructed a new tool that functions at the microlevel scale; specifically, we have created water stress-responsive bacterial biosensors by introducing a fusion between the promoter of the osmotically induced proU operon and gfp, which encodes a green fluorescent protein, into strains of two leaf-associated bacterial species, Pantoea agglomerans and Pseudomonas syringae. In culture, these biosensors respond to limited water availability in a dose-dependent manner. This dose-dependency was observed when the water stress resulted from either high osmolarity (imposed by NaCl or other salts) or from desiccation (imposed by PEG8000). On leaf surfaces, these biosensors exhibited a detectable increase in fluorescence over time as the leaf surfaces were allowed to dry, demonstrating that bacteria on leaves are exposed to water stress. Furthermore, gradients of fluorescence, particularly surrounding the veins, illustrated the regions most susceptible to water stress during the initial stages of drying of a moist leaf surface. To improve the use of these biosensors for quantifying the extent to which leaf-associated bacterial cells and populations are exposed to limited water availability on leaves, a second, constitutively expressed fluorescent marker, rfp encoding a red fluorescent protein, is being introduced into the biosensor cells. Measurements with these biosensors will indicate the importance of water stress in epiphytic colonization processes and in bacterial adaptation to the phyllosphere environment.