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

The aerial leaf surface provides a good model system for understanding how bacterial exposure to environmental stresses influences colonization of a habitat. We examined spatial patterns of leaf colonization by Pantoea agglomerans and Pseudomonas syringae strains that were marked with a constitutively expressed gfp reporter gene encoding a green fluorescent protein (GFP). Using epifluorescence microscopy, we found that, following inoculation onto bean hypocotyls, these strains exhibited distinct patterns of leaf colonization. P. agglomerans cells were visible as individual cells, rather than apparent aggregates, for 6 days as compared to only 1 day for P. syringae. P. agglomerans localized only to surface sites, including the planar surfaces of the epidermal cells, while P. syringae localized quickly to stomatal openings and regions surrounding the glandular trichomes. To evaluate the role of environmental stresses, particularly water stress, in bacterial localization to particular sites, we created water stress-responsive biosensors by introducing a fusion between the promoter of the osmotically induced proU operon and gfp into P. agglomerans and P. syringae. In culture, these biosensors responded to sodium chloride in a dose-dependent manner, as well as responded to both osmotic stress (sodium chloride) and matrix stress (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.