Constitutive expression of the cloned phenol hydroxylase gene(s) from Alcaligenes eutrophus JMP134 and concomitant trichloroethylene oxidation

Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A, R, Darker and Y, Kim, Appl, Environ, Microbiol, 56:1179-1181, 1990), this work represents a purposeful effort to create a constitutive degrader of TCE. Genes responsible for phenol hydroxylase activity were identified by Tn5 transposon mutagenesis, Mutants lacked both phenol hydroxylase and catechol 2,3-dioxygenase activities, Southern blot analysis of total DNA showed that all mutants contained a single copy of Tn5 inserted in the same 11,5-kb EcoRI fragment, Complementation with a cosmid-based gene bank constructed from A, eutrophus AEK101 allowed the isolation of three recombinant cosmids carrying a common 16.8-kb HindIII fragment, Deletion and subcloning analysis localized the genes involved in phenol hydroxylase and catechol 2,3-dioxygenase activities, Partial sequence analysis of regions within the cloned phenol hydroxylase-expressing fragment shows significant homology to the oxygenase and oxidoreductase subunits of toluene-3-monooxygenase from Pseudomonas pickettii, The Tn5-induced phl mutant, carrying a recombinant plasmid expressing the phenol hydroxylase activity, degrades TCE in the absence of induction, Complete removal of TCE (50 mu M) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source, The bacterium removed 200 mu M TCE to below detectable levels within 2 days under noninducing and nonselective conditions.