Financial support: This work was supported by grants 1960262 and 8990004 from FONDECYT-Chile.

Keywords: chloroaromatics, chlorocatechols, metabolic complementation, Ralstonia eutropha, ortho ring cleavage, tfd genes.

Present addresses: # Laboratorio de Microbiología Industrial, Departamento de Ingeniería, Facultad de Ingeniería, Pontificia Universidad Católica de Chile, Av Libertador Bernardo O'Higgins Nº 340, piso 3, Santiago, Chile. Tel: 56 2 686 4053. § Environmental Biotechnology (DGR), Swiss Federal Institute of Technology Laussane (EPFL), CH-1015 Laussane, Switzerland. Tel: 41 21 693 6231. Fax: 41 21 693 4722.

Chloroaromatic pollutants from bleached Kraft pulp mill effluents (BKME) are difficult to degrade, because bacterial strains present in BKME aerobic treatments, only partially degrade these compounds, accumulating the corresponding chlorocatechol intermediates. To improve the catabolic performance of chlorocatechol-accumulating strains, we introduced, by chromosomal insertion, the tfd_ICDEF gene cluster from Ralstonia eutropha JMP134 (pJP4). This gene cluster allows dechlorination and channelling of chlorocatechols into the intermediate metabolism. Two bacterial strains, R. eutropha JMP222 and Pseudomonas putida KT2442, able to produce chlorocatechols from 3-chlorobenzoate (3-CB) were used. Acinetobacter lwoffii RB2 isolated from BKME by its ability to grow on guaiacol as sole carbon source and shown to be able to produce the corresponding chlorocatechols from the BKME pollutants 4-, and 5-chloroguaiacol, was also used. The tfd_ICDEF gene cluster was inserted in the chromosome of these strains using miniTn5-derived vectors that allow expression of the Tfd enzymes driven by the lacI^q/P_trc or tfdR/P_tfd-I regulatory systems, and therefore, responding to the inducers isopropyl-ß-D-thiogalactopyranoside (IPTG) or 3-CB, respectively. Crude extracts of cells from strains JMP222, KT2442 or RB2 engineered with the tfd genes, grown on benzoate and induced with IPTG or 3-CB showed Tfd specific activities of about 15% - 80% of that of the strain JMP134. Dechlorination rates for 3-CB or chloroguaiacols correlated with levels of Tfd enzymes. However, none of the strains containing the chromosomal copy of the tfd_ICDEF cluster grew on monochloroaromatics as sole carbon source. Experiments with BKME aerobic treatment microcosms showed that the catabolic performance of the engineered bacteria was also lower than the wild-type R. eutropha strain JMP134.