CATECHOL 2,3-DIOXYGENASES FUNCTIONAL IN OXYGEN-LIMITED (HYPOXIC) ENVIRONMENTS

We studied the degradation of toluene for bacteria isolated from hypox ic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders, Three Pseudomonas isolates , P. picketti PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS 215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K-m and V-max) for catechol 2,3-dioxygenase (C230), a key shared enzyme o f the toluene-degradative pathway for these strains, acid compared the se parameters with those for the analogous enzymes from archetypal tol uene-degrading pseudomonads which did not show enhanced, nitrate-depen dent toluene degradation, C230 purified from strains W31, PKO1, and CF S215 had a significantly greater sanity for oxygen as well as a signif icantly greater rate of substrate turnover than found for the analogou s enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleot ide and deduced amino acid sequences of C230 from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the s ubfamily of C230s that preferentially cleave monocyclic substrates, Mo reover, deletion analysis of the nucleotide sequence upstream of the t ranslational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of s equences critical for regulated expression of tbuE, including a sequen ce homologous to the ANR-binding site of Pseudomonas aeruginosa PaO. W hen present in ris, this site enhanced expression of tbuE under oxygen -limited conditions, Taken together, these results suggest the occurre nce of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and x ylenes in hypoxic environments. Strain PKO1, which exemplifies this no vel group of microorganisms. compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic paramete rs favorable to function in hypoxic environments, as well as by elevat ing synthesis of such an enzyme in response to oxygen limitation.