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

Given the demonstrated phenol-dependent trichloroethylene (TCE) degradation in Alcaligenes eutrophus JMP134 (A. R. Harker 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 microM) within 24 h was observed in minimal medium containing only 0.05% ethanol as a carbon source. The bacterium removed 200 microM TCE to below detectable levels within 2 days under noninducing and nonselective conditions.