ACTIVITY-DEPENDENT FLUORESCENT LABELING OF BACTERIA THAT DEGRADE TOLUENE VIA TOLUENE 2,3-DIOXYGENASE

Alternative substrates for the toluene 2,3-dioxygenase pathway of seve ral pseudomonads served as enzyme-activity-dependent fluorescent probe s for the bacteria. Phenylacetylene and cinnamonitrile were transforme d to fluorescent and brightly colored products by Pseudomonas putida F l, Pseudomonas fluorescens CFS215, and Barkholderia (Pseudomonas) stra in JS150. Active bacteria transformed phenylacetylene, producing brigh t yellow solutions containing the putative product 2-hydroxy-6-oxo-7-o ctyn-2:4-dienoate. Transformation of cinnamonitrile resulted in bright orange solutions due to accumulation of the putative product 2-hydrox y-6-oxo-8-cyanoocta-2,4,7-trienoate. Chemical and physical properties of the products supported their identification, which indicated that t he first three enzymes of the pathway catalyzed product formation. Phe nylacetylene labeled bacteria with green fluorescence emission; bacter ia were concentrated on black 0.2-mu m-pore-size polycarbonate filters containing polyvinylpyrrolidone (PVP) as a wetting agent. Bacteria la beled with cinnamonitrile were fluorescent orange; labeling was effect ive with bacteria trapped on PVP-free polycarbonate filters. Productio n of the enzymes involved in labeling of P. putida Fl and P. fluoresce ns CFS215 was induced by growth (on arginine) in the presence of tolue ne, cells grown on arginine without toluene were not labeled. Labeling of P. putida Fl by phenylacetylene was inhibited by toluene, indicati ng that the same enzymatic pathway was required for transformations of both substrates. Bacteria expressing other toluene-degrading enzymati c pathways were not fluorescently labeled with phenylacetylene.