We have constructed and characterized bacterial biosensors containing transcriptional fusions that measure the availability of water to bacteria. These fusions involve the promoter of the proU operon (P proU) from Escherichia coli and a reporter gene, either gfp, which encodes a green fluorescent protein, or inaZ, which encodes an outer membrane protein capable of acting as an ice nucleus. These fusions responded in a quantitative manner to water deprivation caused by both osmotic and matric stress and responded to a significant range of water potentials. The PproU- gfp fusion was introduced into a variety of gram-negative and gram-positive bacteria and was found to be osmoresponsive in most of them.;To measure the availability of water to microbes on leaf surfaces, cells of Pantoea agglomerans cells containing PproU -gfp were inoculated onto bean leaves and the plants were exposed to various relative humidity (RH) levels in an environmental chamber. Cells were exposed to an average water potential of -0.4 MPa on bean leaves at all RH levels tested; however, at lower RH levels cells encountered low water potentials faster than at higher RH levels. Based on culture studies, the water potentials encountered by the P. agglomerans cells on leaves were not sufficiently low to kill cells, but were low enough to delay growth and to reduce their growth rate.;To measure the availability of water to microbes in the leaf intercellular spaces during the hypersensitive response (HR) and pathogenesis, Pseudomonas syringae pv. tomato cells containing PproU-inaZ with or without various avr genes were infiltrated into Arabidopsis thaliana leaves. Cells experienced water potentials of -1.3 to -2.3 MPa during HR as compared to only -0.6 to -1.0 MPa during a compatible interaction. The restricted population growth during HR, as compared to the abundant growth during pathogenesis, may have resulted from the absence of available water at the infection site. Supporting this, water potentials in culture equivalent to those in an HR lesion severely restricted bacterial growth, whereas those encountered during the development of disease actually enhanced the growth of the cells.