DEVELOPMENT OF A SITE-SPECIFIC RECOMBINATION-BASED BACTERIAL BIOSENSOR SYSTEM TO REPORT BIOAVAILABLE COMPOUNDS.
Carol Casavant1, Gwyn Beattie2, Gregory Phillips2, and Larry J. Halverson1,2.
Departments of 1Agronomy and 2Microbiology, Iowa State University, Ames, IA USA

We have developed a whole-cell bacterial biosensor system that reports the transient expression of a target promoter by effecting an irreversible, heritable change in the biosensor cell. The target promoter in this study was the promoter (PtbuA1) of the toluene/benzene utilization (tbu) operon of Ralstonia picketiii This promoter is responsive to aromatic compounds. In this system, bioavailable pollutants detected by the sensing organism, Pseudomonas fluorescens strainA506, initiate a site-specific recombination event that results in the permanent and heritable loss of a repressor gene that blocks expression of the reporter gene, gfp. This system consists of a fusion between PtbuA1 and a promoter-less copy of the excisase/integrase gene from the phage l, a fusion between the phage l promoter PL and a promoter-less gfp gene, constitutive expression of the transcriptional activator tbuT, and the use of site-specific recombination to remove a repressor gene (cI) whose gene product tightly represses expression of the PL::gfp reporter gene fusion. Bioavailable aromatic pollutant mediated expression of excisase/integrase leads to the excision of the cI repressor gene, which is flanked by DNA sequences at which the integrase/excisase functions (att sites). Loss of the cI repressor gene then results in loss of the cI repressor protein, which leads to irreversible expression of gfp in the original cell and its progeny. Thus gfp expression serves as a heritable marker of pollutant biovailability and prior expression from the PtbuA1 promoter. The efficacy of this system was tested in culture and in complex environments such as in soil and in the rhizosphere. In culture, 100 % of the A506 biosensor population expressed gfp within 3 hours after exposure to 0.1µM toluene and approx. 80% of the population expressed gfp within 1 hour after exposure. The physiological status of the biosensor cells strongly influenced its responsiveness since the biosensor was less sensitive in stationary than exponential phase of growth. We showed that the biosensor was able to detect available toluene in the barley rhizosphere and that barley roots produce one or more aromatic compounds that function as inducers of the tbu operon. This latter result suggests that the toluene/benzene utilization degradative pathway may have evolved from a pathway involved in the degradation of phenolic compounds produced by plants. This PtbuA1-based biosensor system will facilitate the study of bioavailability in complex habitats where exposure to a pollutant may be significant, but transient. Furthermore, these results demonstrate that this site-specific recombination-based reporter system is an effective system for detecting bacterial exposure to an inducer, even long after the induction event.