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PMID: 8795212 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

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

Applied and environmental microbiology ·Vol. 62 ·No. 9 ·1996-09-00 ·Pages 3227-33

Kim Y, Ayoubi P, Harker AR

Abstract

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.

MeSH Terms
Alcaligenes/genetics DNA Transposable Elements Genes, Bacterial Mixed Function Oxygenases/genetics Oxidation-Reduction Trichloroethylene/metabolism
Chemicals
DNA Transposable Elements Trichloroethylene Mixed Function Oxygenases phenol 2-monooxygenase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kim Y
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater 74078, USA.
Ayoubi P
Harker A R
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Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/aem.62.9.3227-3233.1996
Published
1996-09-00
Pages
3227-33
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC168118
Subset
IM
External Links
PubMed source DOI: 10.1128/aem.62.9.3227-3233.1996 PMC Full Text View journal details
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