The Use of Green Fluorescent Protein For Evaluating the Spatial Distribution of Bacteria on Leaf Surfaces and For Identifying Bacterial Exposure to Water Stress.
CATHERINE A. AXTELL* and GWYN A. BEATTIE
Department of Microbiology, Iowa State University, Ames, IA

Although environmental conditions strongly influence microbial growth and activity, the conditions that microbes actually sense in their natural habitats are poorly understood. The aerial leaf surface provides a good model system for understanding how bacterial exposure to environmental stresses influences colonization of a habitat. To determine spatial patterns of bacterial colonization on leaves, we introduced into strains of Pantoea agglomerans and Pseudomonas syringae a plasmid that constitutively expressed the reporter gene, gfp, which encodes a green fluorescent protein (GFP). Following inoculation onto bean hypocotyls and colonization of leaves, the distribution of these strains was examined using epifluorescence microscopy. Both strains localized in distinct sites, but exhibited distinct patterns of colonization. P. agglomerans cells were present on the planar surfaces of the epidermal cells and were present as individual cells for 6 days as compared to only 1 day for P. syringae. P. syringae localized quickly to stomatal openings and established particularly dense populations in the epidermal junctions radiating from the glandular trichomes. The fact that the two species localized to particular sites, coupled with simultaneous population decreases, suggests that some sites favor growth and/or survival. To identify the environmental conditions sensed by bacteria in various sites on leaves, we are constructing bacterial biosensors that produce GFP in response to a single environmental stress. For example, to create water stress-responsive biosensors, we introduced a fusion between the promoter of the osmotically induced proU operon and gfp into 3 species, Escherichia coli, P. agglomerans, and P. syringae. Based on fluorescence measurements, biosensors of all 3 species were induced by NaCl in a dose-dependent manner. In the future, we will use these bacterial biosensors to evaluate bacterial exposure to water stress in the leaf surface habitat.